· vi the research reported in this thesis was financially supported by a grant from the ministry...

University of Groningen

Characterization of CIC transporter proteinsMoradi, Hossein

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I

University of Groningen Characterization of ClC Transporter Proteins Functional analysis of clc mutants in Arabidopsis thaliana

Hossein Moradi

III

RIJKSUNIVERSITEIT GRONINGEN

Characterization of ClC Transporter Proteins

Functional analysis of clc mutants in Arabidopsis thaliana

PROEFSCHRIFT

ter verkrijging van het doctoraat in de Wiskunde en Natuurwetenschappen aan de Rijksuniversiteit Groningen

op gezag van de Rector Magnificus, dr. F. Zwarts, in het openbaar te verdedigen op

donderdag 21 september 2009 om 11.00 uur

door

Hossein Moradi

geboren op 28 juni 1968 te Sari, Iran

IV

Promotor: Prof. dr. J.T.M. Elzenga Copromotor: Dr. F.C. Lanfermeijer Beoordelingscommissie: Prof. dr. B. van Duijn

Prof. dr. J. Hille Prof. dr. S. Shabala

V

To the twelfth Imam (Mahdi )

VI

The research reported in this thesis was financially supported by a grant from

the Ministry of Science, Research and Thechnology of Islamic Repoblic of Iran,

and were performed in the Laboratory of Plant Physiology, which is a part of the

Center for Ecological and Evolutionary Studies (CEES) of the University of

Groningen, The Netherlands.

Cover design: Farveh Norouzi

Printed: Facilitairbedrijf, RuG, Groningen, The Netherlands

ISBN: 978-90-367-3970-2 (printed version)

ISBN: 978-90-367-3971-9 (digital version)

VII

Contents

Chapter 1 Chloride channels: A general introduction 1

Chapter 2 Generation and characterization of anion channel mutants in 25 Arabidopsis thaliana

Chapter 3 NO3

- and H+ fluxes in Atclcd mutants of Arabidopsis thaliana 43 Chapter 4 Anion channels and root elongation in Arabidopsis thaliana 57

Chapter 5 The role of AtClCa and AtClCd in heavy metal tolerance in 73 Arabidopsis thaliana

Chapter 6 General discussion and conclusion 89 References 93 Summary (English) 107

Summary (Dutch) 109

Summary (Persian) 112

Acknowledgements 114

1

Chapter 1 Chloride Channels: A General Introduction Hossein Moradi1,2 Theo Elzenga1 and Frank Lanfermeijer1

1 Department of Plant Biology, University of Groningen, 9750 AA Haren, The Netherlands 2 Department of Agronomy and Plant breeding, Sari Agricultural Sciences and Natural Resources

University, Iran

Chapter 1

2

INTRODUCTION

The physiology of plants strongly depends on solute and water fluxes across the

cell plasma membrane, the tonoplast and other endomembranes. Among the different

transporter systems involved in transport, ion channels represent a large class with

important roles. These proteins facilitate passive fluxes of ions down their respective

electrochemical gradients. The ClC proteins constitute a family of transmembrane

transporters that either function as anion channel or as H+/anion exchanger. Part of the

ClC family members are anion selective ion channels, which provide passive pores

which allow anions to move according to their electrochemical gradient. The others

are anion/proton antitransporters, which couple the transport of two anions with one

proton. The first member of the ClC family, called ClC-0 was isolated from the

Torpedo marmorata electric organ by expression cloning in Xenopus oocytes (Jentsch

et al., 1990). Figure 1 shows the phylogenetic relationship between different members

of ClC family prokaryotes and eukaryotes. The ClC family has been best

characterized in mammals, in which at least nine members have been identified that

can be grouped in three branches (Jentsch et al., 2002). Several studies have

demonstrated the importance of ClC family in human diseases, such as kidney stones,

muscle disorder myotonia, cystic fibrosis, deafness, and the bone disease osteoporosis

(Jentsch et al., 2005). The first plant member of the ClC family was ClC-Nt1 from

Nicotiana tabacum (Lurin et al., 1996), which was cloned by a PCR-based cDNA

library screening approach (Lurin et al., 1996). ClC proteins in the two model plants,

Arabidopsis thaliana and rice, have been shown to encode anion channels and

transporters involved in nitrate homeostasis. ClC proteins in plants participate in

various physiological processes, such as, osmoregulation, stomatal movement, cell

signaling, nutrient uptake and metal tolerance (Barbier et al., 2000). However,

detailed knowledge on the role of ClC proteins in plant cells is still lacking as a result

of the absence of distinctive effects of knock-outs, the unknown intracellular

localization and the co-operation between the different family members.

Chloride channels: A general introduction

3

Figure 3. Neighbor-Joining Consensus tree of ClC proteins of various kingdoms The tree was calculated using the Geneious program. The branch containing the AtClCe and AtClCf proteins is indicated with “cyanobacteria, mitochondria, chloroplasts”. Arabidopsis thaliana: AtClCa: CAA96057.1; AtClCb: CAA96058.1; AtClCc: CAA96059.1; AtClCd: P92943.2; AtClCe: AAK53390.1; AtClCf: AAK53391.1; AtClCg: P60300.1; Escherichia coli: EcClC1: P37019.2; Homo sapiens: HsClC1: P35523.2; HsClC2: P51788.1; HsClC3: P51790.2; HsClC4: P51793.2; HsClC5: P51795.1; HsClC6: P51797.1; HsClC7: P51798.2; HsClCKa: P51800.1; Neurospora crassa: NcClCx1: EAA33130.2; NcClCx2: EAA28009.2; NcClCx3: EAA28099.2; Nostoc punctiforme: NpClCx1: YP_001868013.1; NpClCx2: BAB73778.1; NpClCx3: YP_001866371.1; NpClCx4: YP_001865245.1; NpClCx5: YP_001865422.1; Saccharomyces cerevisiae: ScClC (GEF1): P37020.1; Salmonella enterica: SeClC1: AAL19167.1; Synechococcus elongatus: SelClCx1: YP_170743.1; SelClCx2: YP_400274.1; SelClCx3: YP_400605.1; Torpedo marmorata: TmClC-0: CAA40078

Transport across cell membranes

The important function of the plasma membrane is to isolate the exterior from the

interior of the cell in order to allow the biochemical processes and preservation of

labile biological molecules. However, in order to facilitate the exchange of substrates,

products and waste this isolation can not be absolute. Because only a very small

number of small lipophilic molecules, like O2 and CO2, can traverse the membrane

unmediated, biological membranes contain various systems that allow the controlled

passage of molecules. These systems are hydrophobic proteins that are inserted in the

Chapter 1

4

membrane and create pores or passageways for all kinds of molecules. These systems

can be divided in two major groups: passive transporters and active transporters.

Passive transport

Transport is considered to be passive when the movement of the solute is solely

driven by the concentration gradient that exists between the interior and the exterior of

the cell. In this case there is always a net flux from the compartment with a high to the

compartment with a low concentration. Subsequently, passive transport can be

divided into simple diffusion and facilitated diffusion. Osmosis, in that sense, does not

differ from solute transport. The only difference is that it concerns water transport.

Osmotic water fluxes are also driven by the differences in concentration. The higher

the concentration of dissolved solutes the lower the concentration of water and, thus,

water moves to areas with a high solute concentration and, thus, a low water

concentration (activity).

Simple diffusion

Simple diffusion through the membrane has only been demonstrated for a few

uncharged lipophilic molecules, like for instance O2, CO2 and NH3. However,

transport can be slow and can not be controlled. Hence, most fluxes of molecules are

facilitated by pores, formed by proteins.

Facilitated diffusion

Routes for facilitated diffusion are created by the insertion of hydrophobic

proteins into the membrane. These proteins have a hydrophobic surface, which allow

interaction with, and thus insertion into, the lipophilic membrane. Internally they

either contain a hydrophilic pore or a hydrophilic pathway, which allows the passage

of the solutes. These pores can be highly specific, in the sense that they allow only

passage of one single type of molecule, for instance potassium channels that only

allow the passage of K+ ions. Another group of ion-specific channels are the chloride

channels, they are named chloride channels because this was the first activity of these

channels detected. However, they are able to mediate fluxes of a few anions (Cl-, Br-,

I-, NO3-). Other pores are less specific and allow the passage of various types of

molecules. Fluxes through the channels continue until equilibrium in concentration is

reached (in the case of uncharged molecules) or if the Nernst potential for the ions


5

transported is established. The Nernst potential considers next to the difference in

concentration also the fact that charges are transported. As soon as for instance K+

flows through a potassium-specific channel it leaves a negative charge behind and

thus a potential difference across the membrane is generated. At a certain moment the

polarization is so large that K+ ions cannot move anymore. The potential at which this

happens is called the Nernst potential for that particular ion.

An important aspect of channel-proteins is that they can be controlled. The

channels can be opened or closed according to the needs of the cell. This phenomenon

is called “gating”. Gating can be controlled by ligands, by membrane potential

(voltage-gated), by post-translational modifications or mechanically. Worth

mentioning in this context is the presence of two CBS domains in the ClC-proteins. It

is suggested that the CBS domains form a sensor that switches transporters between

an inactive and an active state (channels: gating) by interaction of the CBS domains

with the negatively charged membrane surface in response to the ionic strength. This

switching mechanism is an effective means for cells to respond to osmotic shifts,

because an increase in medium osmolality will result in a decrease in cell volume, and

the accompanying increase in cytoplasmic ionic strength will activate the transporter

(Poolman et al., 2006). The presence of these sensors in ClC proteins can be related

with the role of these channels in osmo-regulation, turgor-homeostasis and cell

growth.

Active transport

Cells need to accumulate compounds for different reasons and, thus need to

transport solutes against their concentration gradient. This can be achieved in three

ways. Firstly, the uphill transport of a solute is driven by the release of chemical

energy from the hydrolyzation of ATP or pyrophosphate (PPi), by redox reactions

(respiratory chain) or by light energy (photosynthetic apparatus). Secondly, the uphill

transport of a solute is coupled to the down hill transport of an other solute, and

finally, charged molecules move as a result of membrane potential against their

concentration gradient. The first type of transport is called primary active transport,

the second is a form of secondary active transport and the third is passive transport

down the electro-chemical gradient (but up the chemical gradient).

Chapter 1

6

Primary active transport

While the respiratory chain and the photosynthetic apparatus are special cases,

solute transport is usually energized by the hydrolysis of ATP. Four transport systems

exist which mediate primary active transport of solutes by ATP hydrolysis: P-type

ATPases, V-type ATPases, F0F1-ATPase and ABC-transporters. Important in plant

cell growth are the three primary transporters located in the plasma membrane and

tonoplast. These are the plasma membrane (PM) H+-ATPAse, the tonoplast V-type

H+-ATPase and the tonoplast H+-pyrophosphatase (PPase). Because these primary

transporters generate the proton-motive force across the plasma membrane and

tonoplast, they play an important role in the growth of cells. In that context their

activity is regulated by for instance growth controlling plant hormones like auxins

(Kitamura et al., 1997).

The PM H+-ATPase

The PM H+-ATPase has two roles in the plant cell: firstly it plays a role in

maintance of the cytosolic pH and secondly it generates the proton-motive force

across the PM which is used for the uptake of other solutes. The PM H+-ATPase is a

single-subunit protein and belongs to the P-type ATPases that extrudes H+ from the

cell. This proton pump is able to generate membrane potentials ranging from -120 to -

160 mV (negative inside) and a pH gradient of 1.5 to 3 units (acid outside). The

membrane potential and the pH gradient form the proton-motive force which enables

the uptake of other solutes (Sze et al., 1999).

The Tonoplast H+-ATPase and Tonoplast H+-pyrophosphatase

The tonoplast H+-ATPase is a V-type ATPase and, as such, a multimeric complex

encoded by at least 26 genes (Strompen et al., 2004). The tonoplast H+-pumping

pyrophosphatase (H+-PPase) is single subunit proteins. Both these primary

transporters pump H+ into the vacuole. This action results in a pH gradient and a

membrane potential across the tonoplast. The pH of the vacuole is usually in the range

3-6 while membrane potentials up to 60 mV have been measured (positive in the

vacuole). These two primary pumps generated a proton motive force across the

tonoplast, which is used for the accumulation of solutes into the vacuole. These

solutes can be waste or toxic compounds (Na+), however, the majority of these solutes

are accumulated in order to generate a low water potential necessary for water uptake,


7

turgor and growth. V-type ATPases have also been found in the endoplasmic

reticulum and trans-Golgi network. (Chanson and Taiz., 1985; Strompen et al., 2005

and Dettmer et al., 2005 and 2006), where they play a role in directing the transport

vesicles to their destination.

Secondary active transport

Secondary active transporters or co-transporters couple the uphill transport of

solutes to a downhill transport of another solute. In plants energy is stored in the

proton motive force (PMF) generated by the three major primary proton pumps and

most secondary active transporters use this PMF by coupling the transport of their

solute to the downhill transport of H+. Two distinct types can be distinguished. First

of all there are the symporters, where the transport of the solute is in the same

direction as the co-transported H+. The second type are antiporters in which the

direction of the substrate is opposite to the transport of the H+.

In the large family of ClC membrane proteins, transmembrane movement of Cl-

and NO3- is facilitated by an antiporter mechanism in which a H+ is transported in the

opposite direction (Accardi and Miller 2004; Scheel et al. 2005). A recent

electrophysiological and molecular study demonstrated that ClC homologues are

antiporters in the vacuole of Arabidopsis that, through NO3-/ H+ exchange,

concentrate NO3- in a plant vacuole (De Angeli et al 2006).

Anion transporters

Chloride channel proteins

An important group of anion transporters in plants is the chloride channel (CIC)

family. Since the cloning of first member of the ClC family from the Torpedo electric

organ (ClC-0), these transporter-proteins have been identified in almost all organisms

(Gurnett et al., 1995; Klock et al., 1994). In mammals, ClC proteins form a family of

at least 9 different genes, which can be classified in three subfamilies (Jentsch et al

2005). While more and more individual ClC genes have been identified recently, a

nice synopsis of the presence of this gene family in plants can be obtained from the

complete genome sequencing projects. In the Arabidopsis genome 7 ClC genes are

present (Hechenberger et al 1996). In plants, ClC proteins participate in various

physiological functions, such as, osmoregulation, stomatal movement, cell signaling,

Chapter 1

8

nutritent uptake and metal tolerance (Barbier-Brygoo et al., 2000). Like for all other

organisms, the discussion concerning the real substrates of the ClC proteins in plants

is still continuing. Proteins are designated a chloride channel (ClC) based on the fact

that the cDNA from the prime example, ClC-0, isolated from Torpedo, gave currents

typical for the Torpedo electric organ chloride channel in Xenopus oocytes (Hirono

1987; Gundersen 1984). However, during the last years a dualistic character of these

proteins has surfaced. Some members of this family are indeed functional Cl-

channels, but recently evidence has come forward showing that other members of this

family mediate fluxes of NO3- and, evenmore surprising, in some cases the transport

the anions is coupled to a proton counterflux, which changes the nature of the channel

into that of an antiporter.

Structural organisation of ClC transporters

The Escherichia coli EcClC and Salmonella typhimurium StClC proteins were the

first ClC proteins to be crystallized and provided the second structure of a

transmembrane channel protein (Dutzler et al., 2002; Dutzler et al., 2003). These

studies revealed that the members of the ClC family share a conserved structural

organization, consisting of a transmembrane channel domain and in many cases of

cytoplasmic regulatory domains, like the two cystathionine-β-synthetase domains

(CBS1 and CBS2) at the carboxyl end (see above). EcClC crystallizes, and probably

functions, as a homodimer with each subunit containing an independent ion

translocation pore. The subunits exhibit an ‘antiparallel architecture’: one subunit

contains two structurally related halves spanning the membrane with opposite

orientations (Dutzler 2006; Dutzler et al., 2002, 2003). This topology shows similarity

to other transporter proteins, namely the presence of broken α-helixes and partly

inserted α-helixes and the anti-parallel architecture. A common topology of ClC

proteins has been presented in Dutzler (2006), in which 18 α-helices are recognized,

Chl

orid

e ch

anne

ls: A

gen

eral

intro

duct

ion 9

Prot

ein

TIA

R c

ode

Pres

ence

of s

truct

ural

ele

men

t Pr

edic

ted

Func

tion

Con

firm

ed fu

nctio

n by

GS/

PGIP

Ea G

KEG

Pb 27

0 Glu

b G

XFX

Pb 56

4 Tyrc

AtC

lCa

AT5

G40

890.

2 P

+ +

+ +

H+ /N

O3-

De

Ang

eli e

t al.,

200

6

AtC

lCb

AT3

G27

170.

1 P

+ +

+ +

H+ /N

O3-

AtC

lCc

AT5

G49

890.

1 S

+ +

+ +

H+ /C

l- G

axio

la e

t al.,

199

8; L

v et

al

., 20

09

AtC

lCd

AT5

G26

240.

1 S

+ +

+ +

H+ /C

l- G

axio

la e

t al.,

199

8;

Hec

henb

erge

r et a

l., 1

996;

Lv

et a

l., 2

009

AtC

lCe

AT4

G35

440.

1 -

K to

P

E to

S

F to

Y

- A

-

AtC

lCf

AT1

G55

620.

1 -

K to

P

E to

T

F to

Y

- A

- M

arm

agne

et a

l., 2

007

AtC

lCg

AT5

G33

280.

1 S

E to

A

+ +

+ A

- , no

pH

gat

ing

Tab

le 1

: Stru

ctur

al c

hara

cter

istic

s of t

he A

rabi

dops

is th

alia

na C

lC p

rote

ins a

nd th

eir p

redi

cted

func

tion,

bas

ed u

pon

thes

e ch

arac

teris

tics.

a : The

pre

senc

e of

a p

rolin

e or

a se

rine

at p

ositi

on 2

of t

he m

otif

is in

dica

ted

by a

“P”

or a

n “S

”, re

spec

tivel

y. T

he

abse

nce

of th

e m

otif

in th

e pr

otei

n is

indi

cate

d by

“-“

. b : The

pre

senc

e of

the

exac

t mot

if is

indi

cate

d by

“+”

, If t

he m

otif

is p

rece

nt

in a

mod

ified

form

the

chan

ge is

indi

cate

d by

the

one

lette

r am

ino

acid

cod

e. c : T

he p

rese

nce

or th

e ab

senc

e of

the

tyro

sine

is

indi

cate

d by

“+”

or “

-“, r

espe

ctiv

ely.

Chapter 1

10

of which 17 are fully or partly inserted into the membrane. If this structure can be also

applied to the plant ClC proteins, remains to be seen. At least AtClCa,b,c,d, and g,

which show a high homology with EcClC and contain clearly the conserved

functional domains (GP/SGIP and GK/REPG), might show this topology (Table 1).

AtClCe and f have a lesser homology with the archetype and for instance lack the

GP/SGIP motif (Table 1) and might therefore differ structurally. If plant ClC proteins

function as homodimers, like their bacterial counterparts, also remains to be

determined. Most bacteria contain only one ClC gene, whereas for instance

Arabidopsis contains seven. If some of these plant ClC proteins are targeted to the

same membrane the formation of heterodimers is a possibility.

The ClC transporter family is an interesting group of transporters, as the overall

structural organization of these proteins allow the members either to function as a

channel or a transporter or even as both. This is not an oddity, but a universal property

of possibly all eukaryotic CLC members (Dutzler, 2006, 2007). Therefore, the

molecular architecture of the protein should be able to support both modes of

transport. In the structure of EcClC, but also present in the amino acid sequences of

the Arabidopsis ClC proteins, several essential motives and amino acids have been

recognized. In the crystal structures of the ClC proteins three Cl- binding sites were

recognized (Dutzler et al., 2002; 2003). The first one is, together with other elements,

created by the 564Tyr residue (numbering for AtClCa) and the serine residue of the

motif GSGIP (Figure 2). This site is referred to as the central binding site (Scen). The

internal (close to the cytoplasm) binding site (Sint) is formed by main-chain amide

nitrogen atoms of less conserved amino acid residues (Figure 2). The third binding

site (Sext; Figure 2), which was only recognized after changing glutamate148

(counting in E.coli) to alanine is formed by residues from conserved motifs GK/REGP

and GXFXP (Dutzler et al., 2000). Together these three sites in the channel protein

form the path along which the Cl- ions travel according their electrochemical

potential.

Recently an important observation was made in relations to the NO3- versus Cl-

specificity of the transporters. The Arabidopsis AtClCa protein which is a NO3-

transporter in which the transport of 2 nitrates into the vacuole is tightly coupled to

movement of a proton in the opposite direction, contains, instead of the serine in the

GSGIP motif, a proline. Mutating AtClCa (P to S) and the mammalian ClC-5 (S to P)

at this position, showed the importance of these residues in substrate specificity. In


11

Figure 2a.The gating mechanism of CLC proteins which function as channels (see text) The cartoon displays one monomer. A: Open conformation. B: Closed conformation. C- and CH; deprotonated and protonated carboxyl group, respectively, of the gating glutamate (see text). Sext, Scen and Sint indicate the three anion binding sites. The respective elements forming the binding sites are indicated in the open configuration. Sint is formed by main-chain amide nitrogen atoms.

Figure 2b. Model of the transport mechanism of ClC proteins which function as 2A-/H+ antiporters The cartoon displays one monomer cycling through the different conformations. C- and CH: deprotonated and protonated carboxyl group, respectively, of the gating glutamate (see text). E- and EH: deprotonated and protonated gating glutamate (see text).The transport cycle: Step 1: All three binding site become occupied by an anion (in this case a Cl-). The gating glutamate is protonated and in the open conformation. The proton-donating becomes protonated (can also take place at step 2). Step 2: The

Chapter 1

12

gating glutamate deprotonates and “pushes” the anions through the channel. Two anions leave the channel. Step 3: The channel becomes blocked between Sint and Scen (see figure 3) which prevents back flow of anions. A proton is transferred from the proton-donating glutamate to the gating glutamate and the gates opens and the system returns at step 1.

AtClCa, the P to S mutation resulted in a Cl-/H+ exchange comparable to NO3-/H+

exchange, while in the wild type protein Cl- transport is negligible. The opposite

change in the mammalian ClC-5 protein, which normally transports Cl- tightly

coupled to H+ and NO3- almost uncoupled, resulted in a coupled NO3

- transport

(Bergsdorf et al., 2009). Table 1 shows the distribution of the GS/PGIP variation over

the Arabidopsis CLC proteins.

Two other important residues are the glutamates at positions 203 and 270

(numbering in AtClCa). Glutamate203 is part of the motif GK/REPG and is highly

conserved in the ClC proteins. In Arabidopsis only AtClCg has an alanine at this

position (GKAPG), the other 6 contain this glutamate. In the first structures of the

ClC proteins only two binding sites for chloride were recognized (Sint and Scen)

because of there occupation by chloride ions. The third binding site (Sext) was only

recognized after the respective glutamate in EcClC was mutated, resulting in an

additional halogen anion in the crystal structure (Dutzler et al., 2003). As a

consequence, the gating mechanism of the ClC channels is assumed to be mediated by

this glutamate, which under the proper conditions (pH) mimics a chloride anion and

binds in the Sext binding site and closes the channel. The change to an alanine results

in a channel, which can not be closed (Dutzler et al. 2003; Dutzler, 2006; 2007; Jian et

al., 2004). Recently, also a role of this glutamate in the functioning of the ClC

transporters has been observed. In AtClCa, which in Xenopus shows NO3-/H+

exchange and to a lesser extend Cl-/H+ exchange, mutating 203Glu results in uncoupled

anion conductances, indicating a role of this glutamate in the coupling of the transport

of protons to the anions (Bergsdorf et al., 2009). This effect of this amino acid change

was also observed in other ClC transporters (Accardi and Miller., 2004; Zdebik et al.,

2008).

Changing the 270Glu to an alanine completely abolished the anion currents

mediated by AtClCa in Xenopus-oocytes. However, currents could be restored by the

uncoupling Glu203Ala mutation (Bergsdorf et al., 2009). The idea is that 270Glu,

which is located at the cytoplasmatic site of the membrane, binds protons and hands


13

them over to the gating 203Glu, which results in the coupling of the anion flux to the

proton flux (Accardi et al., 2005; Dutzler, 2007; Lim and Miller, 2009; Zdebik et al.,

2008).

Based on the structural information, given above, predictions can be made about

the function of the Arabidopsis ClC proteins (Table 1). The model described above

can be applied to AtClCa, b, c, d and g resulting in AtClC a and b being NO3-

transporters and AtClCc and d being Cl- transporters. The absence of the equivalent

glutamate residue of 203Glu in AtClCg suggests this might be a channel. However, its

anion preference is difficult to deduce. AtClCa has been shown to function as a

H+/NO3- (De Angeli et al., 2006). AtClCc and d are able to complement the chloride

transporting ClC protein in yeast, GEF1. AtClCa was not able to do so. Those

observations in yeast are in agreement with the role of these proteins as chloride

transporter or nitrate transporter, respectively (Gaziola et al., 1998; Hechenberger et

al., 1996). AtClCe and f, on the other hand, are more difficult to label. They show the

lowest homology with EcClC and the other Arabidopsis ClC proteins. They even lack

some critical residues (for instance the 564Tyr) and motifs (GS/PGIP), hence the

function and role of these two ClC proteins based on their sequence is difficult to

predict.

Figure 1 shows the phylogenic tree containing a considerable set of ClC proteins

from all the major kingdoms and the Arabidopsis proteins. As can be observed, 5 of

the Arabidopsis proteins form their own branch. Only AtClCe and f mingle with ClC

proteins from other kingdoms and more particulary with those from cyanobacteria.

This suggests that these ClC proteins are more related to cyanobacterial proteins

which could be explained by the cyanobacterial origine of the chloroplast and, this

indicates that AtClCe and f are located in the chloroplast.

Calculating a phylogenic tree of a large set of plant CLC proteins resulted in

another picture (Figure 3). In this situation the ClC proteins were organized according

to their characteristics as also used in Table 1. First a large branch could be split off in

which the GS/PGIP motif is absent and a modified GKEGP motif was present (the

lysine was replace by a proline, hence: GPEGP). In this group the proton-donating

glutamate is also absent. This branch contains both AtClCe and f and in combination

with the location of these two Arabidopsis proteins in figure 1 this suggests that the

other plant ClC proteins of this branch are also anion channels which function in

chloroplasts or mitochondria. The absence of the GS/PGIP motif has until now not

Chapter 1

14

Figure 3. Neighbor-Joining Consensus tree of plant ClC proteins The tree was calculated using the Geneious program. Branches are grouped according the presence of elements in the sequence: GP/SGIPE, GKEGP, GXFXP, “glu” indicates the proton-donating glutamate (see text). When the respective element has been striked through this element can not be detected in the protein sequences of the group. Bold and underlined residues indicate differences between this motif with the other groups or the consensus. Arabidopsis thaliana: AtClCa: CAA96057.1; AtClCb: CAA96058.1; AtClCc: CAA96059.1; AtClCd: P92943.2; AtClCe: AAK53390.1; AtClCf: AAK53391.1; AtClCg: P60300.1; Glycine max: GmClC1: AAY43007.1; Medicago truncatula: MtClCx1: ABE91957.1; Nicotiana tabacum: NtClC1: CAA64829.1; NtClC2: AAD29679.1; Oryza sativa: OsClC1: BAB97267.1; OsClC2: BAB97268.1; OsClCx3: NP_001047143.1; OsClCx4: NP_001047955.1; OsClCx5: NP_001062147.1; OsClCx6: NP_001066692.1; OsClCx7: NP_001054061.1; Physcomitrella patens: PpClCx1: EDQ80065.1; PpClCx2: EDQ78881.1; PpClCx3: EDQ52731.1; PpClCx4: EDQ64061.1; PpClCx5: EDQ63773.1; Populus trichocarpa: PtClCx1: EEE85399.1; PtClCx2: EEE77376.1; PtClCx3: EEF09978.1; PtClCx4: EEF01954.1; PtClCx5: EEF10085.1; PtClCx6: EEE99668.1; PtClCx7: EEE84906.1; Ricinus communis: RcClCx1: EEF34561.1; RcClCx2: EEF47977.1; RcClCx3: EEF31629.1; RcClCx4: EEF33157.1; RcClCx5: EEF50918.1; RcClCx6: EEF45376.1; Solanum lycopersicum: SlClCx1: CAC36403.1; Solanum tuberosum: StClCx1: CAA71369.1; Vitis vinifera: VvClCx1: CAO47567.1; VvClCx2: CAO71138.1; VvClCx3: CAO67080.1; VvClCx4: CAO66848.1; VvClCx5: CAO48998.1; VvClCx6: CAO69292.1; VvClCx7: CAO46902.1; Zea mays: ZmClCx1: ACN33881.1; ZmClCx2: AAP04392.2 been implicated with a characteristic of these ClC proteins. It is not known whether

this affects ion-specificity or other transport characteristics. The effect of replacing

the lysine next to the gating glutamate with a proline also is unknown, although an


15

effect on the pKa of the glutamate can be expected and, thus maybe on the

transporters pH-dependence. Hence, these proteins are probably pH-sensitive anion

channels.

A second group, which includes AtClCc, is characterized by the presence of the

two motifs GSGIP and GKEGP and the presence of the proton-donating glutamate.

These proteins are therefore probably H+/Cl- exchangers.

AtClCg is a member of a third group, which is typified by the presence of the

motifs GSGIP and GKAGP. Also the proton-donating glutamate is present in this

branch. As shown in Bergsdorf et al (2009) the engineered combination of the

presence of the proton-donating glutamate and the absence of the gating glutamate in

AtClCa resulted in an uncoupled Cl- and NO3- conductance. Consequently, this branch

probably represents genuine anion channels. However, AtClCa is a H+/NO3-

exchanger, based on the presence of the motif GPGIP which changes to an anion

channel with a higher conductance for Cl- than for NO3- when the proline is changed

to a serine. How the serine in the GSGIP motif in the branch of AtClCg affects the

characteristics of these ClC proteins is unknown.

The final branch, which can be distinguished, is a branch representing H+/NO3-

exchangers. The proteins in this branch contain the GPGIP and GKEGP motifs and

the proton-donating glutamate, which are all features in accordance with a H+/NO3-

exchanger.

Moreover, if one considers the few plant species of which a (almost) complete

genome is available, Vitis vinifera, Oryza sativa, Arabidopsis thaliana and Populus,

all branches of the tree contain at least one protein of these species. This suggests an

early diversification of the ClC proteins in plants and a low redundancy of function

between the members of the different branches.

Tissue and intracellular localization

An important indication for the function of proteins is their functional localization.

In this respect both tissue and intracellular localization are important. Those two

levels of localization are mainly regulated in two different ways. Whereas tissue-

specific expression and developmental stage-specific expression are controlled at the

gene level, intracellular localization is controlled by sorting peptides present in the

protein. However, the nature of the translation product (different splice forms or

alternative translation initiation), controlled at gene or RNA level, can also affect

Chapter 1

16

intracellular localization (Millar et al., 2009). Tissue expression is studied by gene-

expression studies or by promoter fusions. Lv et al., (2009) made a thorough analysis

of tissue-specific AtClC-gene expression by RT-PCR and promoter-driven GUS

expression. In their RT-PCR experiments ubiquitous expression of all ClC genes

throughout the plant was observed with only small variations in the level of

expression amongst the tissues. Such an expression profile suggests that the various

ClC proteins have distinct individual functions and roles and have little or no

redundancy. Interesting in this context, are the more or less inverse expression

profiles of AtClCe and f. While AtClCe is more expressed in leaf, flower and silique,

AtClCf is more expressed in root and stem. As suggested above these two proteins are

probably functioning in either the chloroplast or mitochondrion.

The histochemical study of Lv et al. (2009) also demonstrated that ClC members

have individual functions. There expression patterns overlapped, but they had also

their differences. The largest differences were observed between AtClCa, b, c, d and

g, on the one hand, and AtClCe and f on the other. The temporal and spatial

distribution of AtClCe and f suggest a relation with the presence of functional

chloroplasts. No evident expression was observed for these genes in the root.

Moreover, it has been shown that photosynthesis is disturbed in mutants of AtClCe

(Marmagne et al., 2007).

Another important issue is the subcellular localization of the ClC proteins.

Predictions can be made using the Aramemnon web-based prediction tool (Table 2)

but recently several studies using fusions of the AtClC proteins with fluorescent

passenger proteins (FP) like Green Fluorescent Protein derivatives or Discosoma sp.

Red (DsRed) has been used (De Angeli et al., 2006; Fecht-Bartenbach et al., 2007; Lv

et al., 2009; Marmage et al., 2007) (Table 2). Also, a few ClC proteins of Glycine

max and Oryza sativa have been localized using fusions to fluorescent markers (Li et

al., 2006; Nakamura et al., 2006). However, as Moore and Murphy (2009) state:

“Determining protein localization inevitably is an exercise in imperfection.” They

(Moore and Murphy, 2009) and Millar et al. (2009) discuss the state of the art of

intracellular protein localization, summarize the strengths and weaknesses of the

employed protocols and give guidelines for validation of the localization of proteins.

Amongst the issues they raise are the use of strong, heterologous promoters to

generate aesthetically pleasing images and the positioning of the fluorescent

passenger proteins in an construct. Another important feature, which increases the risk

Chl

orid

e ch

anne

ls: A

gen

eral

intro

duct

ion 17

Prot

ein

Ara

mem

non

pred

ictio

n Ex

perim

enta

lly

dete

rmin

ed

Prom

oter

a Lo

catio

n FP

b R

efer

ence

AtC

lCa

stro

ngly

secr

etor

y.pa

thw

ay

tono

plas

t 35

S 35

S C

C

D

e A

ngel

i et a

l., 2

006

Lv e

t al.,

200

9 A

tClC

b

wea

kly

secr

etor

y.pa

thw

ay

tono

plas

t 35

S C

Lv

et a

l., 2

009

AtC

lCc

w

eakl

y se

cret

ory

path

way

to

nopl

ast

35S

C

Lv e

t al.,

200

9

AtC

lCd

secr

etor

y pa

thw

ay

golg

i 35

S 35

S C

C

Fe

cht-B

arte

nbac

h et

al.,

200

7 Lv

et a

l., 2

009

AtC

lCe

mito

chon

drio

n>ch

loro

plas

t> se

cret

ory

path

way

th

ylak

oid

35S

35S

C

C

Mar

mag

e et

al.,

200

7 Lv

et a

l., 2

009

AtC

lCf

mito

chon

drio

n >

secr

etor

y pa

thw

ay

golg

i 35

S 35

S C

C

M

arm

age

et a

l., 2

007

Lv e

t al.,

200

9 A

tClC

g w

eakl

y se

cret

ory

path

way

to

nopl

ast

35S

C

Lv

et a

l., 2

009

Tab

le 2

: Pre

dict

ed a

nd e

xper

imen

tally

det

erm

ined

loca

lizat

ion

of th

e Ar

abid

opsi

s tha

liana

ClC

pro

tein

s a : p

rom

oter

of t

he fu

sion

pro

tein

of t

he C

lC a

nd fl

uore

scen

t pro

tein

, 35S

: Cau

liflo

wer

Mos

aic

Viru

s 35S

pro

mot

er; b : l

ocat

ion

of th

e flu

ores

cent

pr

otei

n, C

: C-te

rmin

al.

Chapter 1

18

of creating artifacts, is the routing of most proteins through various compartments

before they reach their destination. This movement requires saturable transport and

signaling systems, which can result in missorting (Moore and Murphy, 2009),

especially in the case of over-expression. Moreover, during trafficking the various

stations passed could have different amounts of the trafficking proteins. As a result

higher protein amounts can be present at the intermediate stations and the

fluorescence at these locations could outshine the fluorescence of the protein at the

final destination. Even alternative locations, like the tonoplast or the plasma

membrane could be reached due to congestion of the original route (Moore and

Murphy, 2009). Hence, it can be asserted that in the case of membrane proteins, like

ClC proteins, these artifacts are probable to occur. Membrane proteins have a lower

degree of freedom and have to traffic via membranes.

If we consider the guidelines for validation of the location of proteins as suggested

by Millar et al. (2009), the experiments performed in order to determine the location

of the ClC proteins are not optimal. Some of the major concerns are: 1) all studies use

the Cauliflower Mosaic Virus 35S promoter, which results in an uncharacteristically

high expression, presenting for membrane proteins an even larger ‘congestion’

problem, 2) in those studies the fluorescent passenger protein is attached to only one

location in the protein. In the studies with the Arabidopsis, Glycine ClC proteins the

FPs were all fused to the C-terminus of the proteins (De Angeli et al., 2006; Fecht-

Bartenbach et al., 2007; Li et al., 2006; Lv et al., 2009; Marmage et al., 2007). In the

studies with the Oryza proteins the FPs were fused to the N-terminus (Nakamura et

al., 2006). Although the results are in agreement with the ideas of the function of the

ClC protein, this means care must still be taken with the interpretation of the recent

fluorecence data on the localization of the ClC proteins.

For example, the Glycine max ClC1 protein was placed in the tonoplast because of

its co-localization with GmNHX1 (Li et al., 2006). NHX1 is an established tonoplast

protein. However, in this study both proteins were visualized by the use of the strong

Cauliflower Mosaic Virus 35S promoter. Although both proteins display a similar

localization, we are doubtful about the result that shows a tonoplast localization.

Apparently, both proteins accumulate in endomembrane vesicles which either

outshine the proteins in the tonoplast, or are the result of congestion of the transport

systems. Lurin et al. (2000) studied the localization of NtClC1 using fractionation and

Western-blotting and concluded that this protein localizes to mitochondria. The


19

closest homologue of NtClC1 in Arabidopsis is AtClCc, which is experimentally

located in the tonoplast (Table 2). In spinach a ClC protein was found in the outer

envelope of chloroplast by mass-spectrometry and membrane fractionation (Teardo et

al., 2005). This spinach protein gave three peptides that had sequences identical to the

partial sequences of the AtClCf protein. However, the AtClCf protein is

experimentally located in the Golgi membrane, but predicted to be targeted to the

mitochondria (Table 2).

Differences between patch-clamp and molecular studies

Electro-physiological studies have been performed on plants for at least 60 years.

In the early years only membrane potentials could be measured by impalement of

electrodes into cells and tissues. This is a technique that only allows a general study

of the behavior of the membrane potential of plant cells upon varying conditions.

Presently the high-resolution electro-physiological method, the patch-clamp

technique, allows the study of single conductances in membranes. However, both

patch clamp and the impalement of electrodes are invasive techniques, which require

isolation of cells or protoplasts or wounding of the tissue. Especially, the patch clamp

technique revealed an enormous number of ion-conductances present in plasma

membrane and tonoplast. However, the matching of conductances with proteins and

their corresponding genes is a laborious process. Forward genetics appears difficult,

starting from a current and trying to find a protein, which is responsible for the

current. However, reverse genetics has proved useful in identifying the transporter

proteins, responsible for the conductances observed by patch clamp. A nice example

of such a study is the characterization of the AtClCa protein in the tonoplast of

Arabidopsis thaliana (De Angeli et al., 2006). In this study it is shown that in two

independent knock-out plant lines, in which AtClCa is absent, a certain nitrate current

could no longer be observed in the tonoplast, indisputably matching the nitrate

conductance to the AtClCa protein. Recently, a new electrophysiological technique

has been developed. The Micro-Electrode Ion Flux Estimation (MIFE) technique

allows the noninvasive and simultaneous monitoring of different ion fluxes from

intact tissues with a high spatial and temporal resolution (Shabala et al., 1997;

Newman, 2001; Tegg et al., 2005; Vreeburg et al., 2005; Lanfermeijer et al., 2008).

Without damaging the tissue this technique is able to detect changes in fluxes of

Chapter 1

20

various ions by the use of ion-specific electrodes. However, no studies with this

technique on ClC proteins are known to us.

The physiology of anions

The most abundant anions in plants are nitrate, chloride, sulfate, phosphate and

malate. Carbonate, despite its low concentration, compared with other inorganic

anions, occupies a particular status, as it plays a role in intracellular pH regulation

and is the major carbon input for photosynthesis (Barbier et al., 2000). Most anions

have important metabolic functions and most can be accumulated in the vacuole. In

plant cells relative concentrations of anions vary, depending on the tissue and

physiological and environmental parameters. In plant cells, the highest anion

concentrations are found in the vacuole, while cytosolic levels are maintained in the

millimolar range. Of these, the inorganic ions have to be taken up from the

environment. In higher plants the root system is responsible for the uptake of nutrients

and, thus, most inorganic anions. Subsequently, the anions (and their counter cations)

are transported to the shoot by the transpiration stream. Although, most of the

transport by the transpiration stream can be apoplastic and does not need passage of

membranes, at least at the Casparian strips in the roots the ions have to enter the

symplast. Hence, they have to pass the plasma membrane at least twice. Anion

channels have been reported in the xylem parenchyma cells of barley roots (Wegner

and Raschke., 1994; Kohler and Raschke. 2000; Kohler et al. 2002), root stellar cells

of maize (Gilliham and Tester. 2005) and Arabidopsis root pericycle cells (Kiegle et

al. 2000). Kohler and Paschke (2000) identified fast and slow activating anion

channels in barley xylem parenchyma cells. After the anions have arrived at the sink

tissue (growing leaves, fruits, etc) they have to enter cells. It is hypothesized that the

influx of chloride occurs via H+ /anion symporters or OH- /anion antiporters (Zeiger et

al., 1978).

Anion fluxes in the plant cells

The different cell compartments require all their specific concentrations of

metabolites and minerals. These concentrations are all maintained by transport

systems, which are energized by ion-gradients and potential differences, generated by

the primary pumps. Anion fluxes play an important role in these processes. First of all


21

in the generation of the proton motive force. They relieve the membrane potential

generated as a consequence of the transmembrane transport of protons. When for each

proton moved an anion is transported in the same direction the membrane protential

does not rise so drastically. This allows more protons to be transported and the proton

gradient to become larger than in the absence of anion fluxes. Although the driving

force on protons is thereby reduced, the power for transport of solutes coupled to

protons is increased. This is the so called shunt-function of anion fluxes.

The second function of anion fluxes is related to the role of anions as osmotically

active solutes. The accumulation of anions and their counter ions in cells drives the

uptake of water into the cells and, subsequently, the generation of cell turgor. Cell

turgor, in its turn drive cell expansion and cell growth.

Their role in water movement and turgor regulation makes anion fluxes also

important in the opening and closing of stomata. Stomata are microscopic pores in the

aerial parts of the plant, which provide a passageway for CO2, that is needed for

photosynthesis, to enter the leaf. Guard cells surround the pore and the swelling and

shrinking of these cells modulate stomatal pore size by coordinating responses to

environmental and physiological factors, including light, temperature, Ca2+, and the

plant hormone abscisic acid. During stomatal opening and closing, chloride and

malate are the major anionic species involved in turgor generation for opening. It has

been known for several decades that guard cells can take up chloride ions during

stomatal opening, but the molecular mechanism of that is still not fully understood.

The role of pH in plant cell growth

Many different physiological events in plant cells are regulated by changes in pH

or depend on proton gradients. In plant cells pH is well characterized as a regulator of

processes, such as modulation of Ca+2 signaling, protein synthesis, and enzyme

activity. In plant cells, according to the plant species and the technique of

measurement used, cytosolic pH values in resting conditions are between 6.8-7.9

(Guern, 1991). In response to osmotic stress and hormone treatment, the cytosolic and

apoplastic pH both fluctuate (Guern., 1991; Tretyn et al., 1991; Nuhse et al., 2000). In

plant cells, the PM H+-ATPase is the primary active transport system and mainly

responsible for generating the membrane potential and the proton gradient and

maintaining the cytosolic pH (Assman and Haubrick., 1996). It is also known that

changes in cytosolic pH can act as a second messenger in plant cells (For review, see

Chapter 1

22

Felle, 1989; Guern et al., 1992; Zimmermann et al., 1999). According to the acid

growth theory (Rayle et al., 1970; Cleland, 1971; Hager et al., 1971) , low pH induces

rapid cell wall loosening and cell elongation. In pea leafs the increased extrusion of

protons by the activated PM H+-ATPase results in the enlargement of the pH gradient

and the hyperpolarization of membrane potential across the plasma membrane, which

result in an increase of the proton-motive force. This stimulates the uptake of nutrients

and osmotically active solutes. Subsequently, water is also absorbed as a result of

osmosis and cell turgor increases (Staal et al., 1994). The extracellularly located

expansins react to the acidification of the cell wall by activation of their cellulose and

hemicellulose degrading properties (Cosgrove, 1998), while Ca2+-pectin cross-links

are broken as a result of displacement of the Ca2+ by H+ (Proseus & Boyer, 2006; den

Os et al., 2007). Both these reactions increase the cell wall elasticity. The increases in

turgor and cell wall elasticity result in cell expansion. But also in roots the elongation

is regulated by acid growth phenomena (Edwards and Scott., 1974; Buntemeyer et al,

1998; Peters and felle, 1999). In Arabidopsis root, changes in root cap pH are required

for the gravitropism (Fasano et al 2001).

Anion and cation channels play an important role in this growth process. The

movement of cations in the opposite direction or an anions in the same direction as the

proton flux, can aid in the generation of the proton gradient (the shunt function; see

above) and increase the extracellular acidification. Changes in the activity of ion

channels can therefore result in changes in the membrane potential and in the pH

gradient (Johannes et al., 1998) and therefore the ability of cells to grow. Secondly,

anions are used as osmotics and a change in the transport capacity of these solutes can

affect growth. The importance of anion fluxes is demonstrated by the fact that in

AtClCd knock out mutants root growth is reduced compared to wildtype at a slightly

alkaline pH of the growth medium (Fecht-Bartenbach et al).

Perspectives

Although their importance in processes like pH homeostasis, growth, abiotic and

biotic stress resistance, osmotic acclimation, nutrient uptake and transport has been

amply demonstrated, the physiological characterization and the knowledge of the

position of ClC proteins in the complex network of membrane transport and solute

fluxes is still incomplete. Mutant analysis, combined with detailed physiological


23

studies can provide us with much of the data necessary to fill these gaps in our

understanding. In this study we used knock-out mutants to elucidate the role of

members of the AtClC transporter family with the use of the MIFE technique. In this

thesis the role of AtClCa and AtClCd in pH homeostasis and metal-tolerance has been

demonstrated

25

Chapter 2 Generation and characterization of anion channel mutants

in Arabidopsis thaliana

Hossein Moradi1,2, Theo Elzenga1 and Frank Lanfermeijer1

1 Department of Plant Biology, University of Groningen, 9750 AA Haren, the Netherlands 2 Department of Agronomy and Plant breeding, Sari Agricultural Sciences and Natural Resources

University (SANRU), Sari, Iran

Chapter 2

26

ABSTRACT

The chloride (Cl-) and nitrate (NO3-) anions are major inorganic constituents

of plant cells. These anions play a role in turgor maintance, signaling (Cl-) and

metabolism (NO3-). An important group of anion transporters are the Chloride

Channel (ClC) proteins, which facilitate the transport of Cl- and NO3- ions. These

proteins are reported in all plant cell membranes and appear to be involved in

various physiological processes such as the control of stomata movements, xylem

loading and plant pathogen interactions.

To study the role of these ClC proteins in plants we obtained three T-DNA

insertion lines for the AtClCa, b, and d genes. In order to characterize the ability

of these genes to complement each other we generated the three double and the

single triple mutants.

Seed germination on media with supplemented with different concentrations

of NaCl, CholineChloride, KCl and KNO3 did not show a difference between the

different genotypes. The Micro-Electrode Ion Flux Estimation technique was

used to monitor and characterize H+ and Cl- fluxes from intact leaf tissue and the

changes in these fluxes induced by changes in the external NaCl concentration.

Although NaCl induces a transient change in the fluxes of chloride and protons,

the responses of the mutant plants were not distinguishable from wild type. Also

morphologically the mutant plants were similar to the wild type. However, when

the pH of the growth medium was increased from 5.8 to 6.2 root growth in

mutant lines, Atclca and Atclcd, the double mutant Atclcad and triple mutant

Atclcabd was reduced.

Generation and characterization of anion channel mutants

27

INTRODUCTION

Although chloride concentrations in plants are considerable, usually the

compound is considered to be an essential micro-nutrient (Broyer et al. 1954).

Chloride is essential for the optimal activity of several enzymes. In photosynthesis

chloride plays a role in the water splitting complex. Chloride also plays an important

role in osmoregulation and growth (Barbier-Brygoo et al. 2000). Cell expansion is

driven by turgor pressure which results from the osmotically driven uptake of water

into the cell. Water enters the cells due to the high concentrations of potassium and

anions, like chloride and malate. During rapid growth, chloride accumulates in the

vacuole of plants cells, where concentrations up to 40 mM can be measured (Barbier-

Brygoo et al., 2000). A specialized, but closely related, role is the involvement of

chloride in the opening and closing of stomata. Stomata open as a result of an increase

in turgor. This turgor increase results from the uptake of water into the guard cells.

The continuous redistribution of chloride between the guard cells and the surrounding

tissue upon changing conditions, is one of the mechanisms controlling the opening

and closing of the stomata (Pandey et al 2007). Another role of chloride is associated

with the negative charge of the ion. Transport of anions across a membrane can

compensate for the currents, which result from transport of positive ions (like

protons), thereby allowing a steeper concentration gradient of these cations across the

membrane. In this context a role of chloride has been demonstrated in proton transport

by the plasma membrane H+- ATPase and the vacuolar V-type ATPase (Felle 1994).

Other transport processes were chloride is either directly of indirectly involved are for

instance Na+/H+ and HCO3-/ Cl- exchange (Jentsch et al 2002).

Mechanistically, at low external concentrations, Cl- can be taken up via a H+/ Cl- co-

transporter, while at high Cl- concentrations the anion might enter passively through

anion channels. Active chloride uptake and transport by roots has been demonstrated

(reviewed by White and Broadley, 2001) and also Cl- transport to the shoot is

controlled by the roots (Sauer, 1968; Downton, 1977; Storey et al., 2003). It is

unknown which systems actually mediate Cl- uptake into the symplast of the root.

Considering the concentration gradient, which exists between the apoplast and the

symplast, the systems involved should be active transporters. Only under high saline

conditions the concentration gradient might be in favor of passive uptake of Cl-

mediated by chloride-specific channels or more non-specific anion channels.

Chapter 2

28

An important group of anion transporting proteins is the Chloride Channel (ClC)

proteins. Seven ClC genes have been identified in the Arabidopsis genome. ClC genes

are present in most organisms: their presence has been shown in fungi, bacteria,

animals, and plants. The ClC protein family has been best characterized in mammals,

in which nine different ClC members are present. These proteins can be divided into

three groups based on their localization in the plasma membrane or intracellular

membranes (Jentsch et al., 2002). The 3D structure of these proteins has been solved

(Dutler et al., 2002; Dutzler et al., 2003) which allows a detailed study of the

structure-function relationship in these proteins. In Arabidopsis the seven members

can be divided into two groups (Lv et al., 2009). This division is based on their

molecular structure and seems to be related to their mode of action. The seven

proteins have been demonstrated as a group to be present in cell membranes where the

different members have their own specific localization (De Angeli et al., 2007;

Marmagne et al., 2007; Lv et al., 2009). They are involved in turgor and

osmoregulation, membrane potential control, vesicle trafficking, nutrient transport,

stomatal movement, and resistance to heavy metals and salt stress (Barbier-Brygoo et

al., 2000; Skerett and Tyerman., 1994; Tyerman et al., 1997).

Despite the key role of chloride in various processes, little is known about the role

of the ClC proteins in Cl- homeostasis and, for instance, salinity stress. Moreover,

interaction and coordination of the activities of the seven ClC proteins can be

expected. In order to address these issues we set out to isolate mutants of ClC genes.

We have identified three Arabidopsis mutants lines which carry a T-DNA insert in the

AtClCa, AtClCb, and AtClCd genes, respectively. We show the absence of transcripts

of these genes in the mutant plants and made the three double mutants and the triple

mutants and performed a preliminary evaluation of those six mutant lines. Under the

conditions we used no obvious phenotypes were observed for the single mutants. We

could only observe a phenotype in the double and triple mutants containing insertions

in both the AtClCa, and AtClCd genes when the plants were grown at a pH higher

than 5.8.


29

MATERIALS AND METHODS

Plant material and growth conditions

Seeds of Arabidopsis T-DNA insertion lines were obtained from NASC (AtClCa:

WiscDsLox477-480I4; AtClCb: SALK_ 27349; AtClCd: SALK_42895). For in vitro

growth experiments, seeds were surface sterilized with gaseous chlorine (derived from

acidified sodium hypochlorite) and sown on a half-strength Murashing and Skoog

medium (MS/2), supplemented with 1% w/v sucrose and buffered with 10 mM MES-

Tris, pH 5.8 and with 0.8% w/v of agar. For the selection of the mutants 50 μg/ml

kanamycin was added. The dishes were sealed with surgery tape (3M). For the growth

analysis the proper salts and additives were added or the pH was adjusted accordingly.

For growth on soil, seeds were surface sterilized and sown in pots contain an

organic-rich soil (TULIP PROFI No.4, BOGRO B.V., Hardenberg, The Netherlands).

All dishes and pots, were incubated in the dark at 4oC for 3 days. Subsequently, the

dishes were transferred to growth chamber set at a 16h/8h light light/dark cycle, 20±2

oC temperature at 72% relative humidity while the pots were transferred to the

greenhouse at 20±1 oC during the 16 h day period and 18±1 oC during the night period

at 72% relative humidity and with supplementary light when necessary, or to the

growth chamber with the conditions as described above.

Screening for T-DNA insertion mutants

The T-DNA insertion disrupting AtClCb and AtClCd were identified in the

database at the SALK Institute Genome Analysis Laboratory (Salk-027349 for

AtClCb ,Salk-042895 for AtClCd) and Wiscd Siox 477-480i4 was identified in the

WiscDsLox T-DNA collection for AtClCa. To obtain homozygous mutants lines,

resistance to kanamycin was checked and PCR-based screens with the respective

primers for each T-DNA were performed according to Salk and Wisc protocols (Table

1).

To generate double mutants, single mutant plants were crossed to each other. The

genotypes of F1 plants were checked using a PCR-based screen with respective

primers for each gene (Table 1). All F1 double mutants (Atclcab, Atclcad, and

Atclcbd) were allowed to self-pollinate. Seeds were sown on plates. Plants were

subsequently checked again with the primer sets of the respective T-DNAs (Table 1)

and segregated in the F2 population progeny in a 1:15 ratio. A triple mutant was

Chapter 2

30

created by crossing between two double mutants Atclcad and Atclcbd. F1 progeny

from this cross were allowed to self-pollinate. Seeds were sown and the progeny was

checked and analyzed for their genotypes (Figure 1).

Figure 1. The T-DNA insertion in the three single mutant parental lines Left: The position of the T-DNA insertions in AtClCa, AtClCb and AtClCd genes. Right: PCR-based markers for the presence and absence of the T-DNA insertions. Lane 1: Homozygous wild type genotype, 2: Heterozygous genotype 3: Homozygous T-DNA insertion genotype. Indicated on the left are the sizes of the MW markers in the marker lane and on the right the gene studied is indicated.

Reverse Transcript PCR

Total RNA from shoots and roots was isolated according to Chomczynski

et al. (1997) using TRIZOL reagent (Invitrogen). First-strand cDNA synthesis

on 3μg of total RNA was done using reverse transcriptase (Fermentas) and a

Oligo(dT) primer according to the suppliers manual. Two microliters from the total 20

μl volume of cDNA was used for PCR amplification using polymerase in a 50 μL

reaction volume according to the following program: 94°C for 2 min, then 32 (AtClCa

and AtClCd) or 35 (AtClCb) cycles consisting of 94°C for 15 s; 54°C for 30 s (AtClCa

and AtClCd), 45°C for 30 s (AtClCb) ;72°C for 1 min and 72°C for 5 min . Table 1

shows the RNA-specific primers for the ClC genes and the control gene, tubelin.

Ion flux measurement in MIFE technique

Net fluxes of H+ and Cl- from leaves were measured using H+- and Cl--selective

microelectrodes with the MIFE technique (Shabala et al., 1997; Newman, 2001;

Gen

erat

ion

and

char

acte

rizat

ion

of a

nion

cha

nnel

mut

ants

31

Targ

et

Left

prim

er

Rig

ht p

rimer

Le

ft B

orde

r prim

er

T-D

NA

inse

rtion

AtC

lCa

5’-C

CA

GA

TAA

ATC

TTC

AC

TTTC

TGA

TGG

5’

-TG

TCA

ATG

CC

ATT

AA

GG

TAA

GC

5’

-GC

GTG

GA

CC

GC

TTG

CTG

CA

AC

T

AtC

lCb

5’-G

GA

GTT

CTG

TAG

CC

CC

AG

TTG

5’

-GTA

ATC

GG

TGG

AA

TTC

TTG

GG

5’

-TG

GTT

CA

CG

TAG

TGG

GC

CA

TCG

AtC

lCd

5’-G

GA

AC

TGG

ATT

AG

CTG

CTG

TG

5’-C

CTA

CC

ATG

ATG

TGA

CC

TCC

TC

5’-T

GG

TTC

AC

GTA

GTG

GG

CC

ATC

G

Gen

e ex

pres

sion

ana

lysi

s

AtC

lCa

5'-A

CTG

CA

TTTT

GG

GTC

TTA

5'

- GA

AA

TGC

TTG

TAG

AA

TGTT

A

AtC

lCb

5'-A

CTG

CA

TCTT

GG

GG

CTT

T 5'

-AG

GC

TTG

TAG

AA

TGTT

GTA

T

AtC

lCd

5’-T

TTA

CA

CA

TTA

GC

TGTA

G

5’-T

TGA

CTG

TTG

GA

GTT

CC

AC

T

β-Tu

belin

5’

-GA

GC

CTT

AC

AA

CG

CTA

CTC

TGTC

TGTC

5'

- AC

AC

CA

GA

CA

TAG

AG

CA

GA

AA

TCA

AG

Tab

le 1

: . T

he P

CR

Prim

ers f

or T

-DN

A c

onfir

mat

ion

and

gene

exp

ress

ion

anal

ysis

, use

d in

this

stud

y.

Chapter 2

32

Lanfermeijer et al., 2008). Microelectrodes were pulled from borosilicate glass capillaries

(GC150-10; Harvard Apparatus) and silanized with tributylchlorosilane (Fluka 90974). The

H+- selective electrodes were back filled with 15 mM of NaCl plus 40 mM of KH2PO4 and

front filled with Hydrogen Ionophore II (Cocktail A; Fluka 95297). The Cl--selective

electrodes were back filled with 500 mM of KCl adjusted to pH 6 with NaOH and front

filled with Cl− (chloride ionophore I, cocktail A, Fluka 24902). The response of the

electrode was typically 48 mV/decade. This somewhat low response voltage was always

observed and is considered to be the result of a small leakage of ions in the tip of the

electrode. Apparently, the sealing between the silanized glass and the ionophore mix was

not optimal. To avoid potassium ion leakage from the reference electrode, the reference

electrode was placed in a compartment different from the measuring chamber. The two

compartments were electrically connected via a salt bridge, which consisted of 300 mM

(NH4)2SO4 in 2% (w/v) agar.

Leaf material was immobilized on a glass capillary using grease (consisting of 49%

petroleum jelly, 34% bee wax, and 17% lanoline) with the abaxial epidermisless side

exposed to the solution and placed in a measuring chamber with a transparent bottom. The

chamber was filled with 1 ml of basic measuring solution (BMS; 1 mM KCl plus 0.5 mM

CaCl2, pH 5.8), submerging the leaf material. The whole chamber was placed on a Nikon

TMS inverted microscope. The ion-selective microelectrodes were mounted at an angle

between 30° and 40° with the horizontal in a holder (MMT-5; Narishige) on a three-way

piezo-controlled micromanipulator (PCT; Luigs and Neumann) driven by a computer-

controlled motor (MO61-CE08; Superior Electric). The electrodes were positioned 10 μm

from the surface of the tissue. During measurements, the distances between the tissue and

the electrodes were changed from 10 to 50 μm at a frequency of 0.1 Hz. The chemical

activities of H+ and Cl- in solution were continuously recorded at the two distances from the

tissue, and from these data, net H+ and Cl- fluxes were calculated according to Newman

(2001). Whereas positioning the material in the MIFE apparatus was performed under light

conditions (150 μmol·m-2·s-1), the measurements were performed in the dark at ambient

room temperature.


33

RESULTS

Isolation of homozygous knockout lines with PCR-based screen

The obtained three single homozygous T-DNA insertion lines in Arabidopsis

ecotype Columbia in AtClCa, AtClCd and AtClCb were used to generate the three

double and the one triple mutant combinations. The exact locations of the T-DNA

insertions in the loci At5g40890, At3g27170 and At5g26240 were obtained from the

TAIR database (http://www.Arabidopsis.org) (Figure 1a). The correct genetic

conformation of the three parental single mutant lines was confirmed by PCR (Figure

1b).

Tissue specific expression of the AtClCa, b, and d genes

Transcripts of AtClCa, and d could be detected in both the shoot and the root.

Transcripts of AtClCb could only be detected in the root (Figure 2).

In the T-DNA insertion lines only transcripts of the ClC genes, which were not

disrupted, could be detected (Figure 3).

Figure 2. Semi-quantative expression of AtClCa, AtClCb and AtClCd in different plant tissues. Shown are the amounts of PCR product resulting from 32 (AtClCa and AtClCd) or 35 (AtClCb) PCR cycles. The Tubulin transcript levels are shown as a loading control. The used primers for the respective genes are shown in Table 1.

Figure 3. The absence of expression of the AtClCa, AtClCb and AtClCd genes in their respective T-DNA insertion lines. The three genotypes were analysed using the primers shown in table 1. The Tubulin transcript levels are shown as a loading control.

Chapter 2

34

Ion fluxes

The MIFE technique allows monitoring of ion fluxes in and out of almost intact

tissues. We explored the potential of this technique to study NaCl induced changes in

ion fluxes in leaf tissue. We focused on H+ and Cl- fluxes because the potential role of

the so-called ClC proteins in Cl- fluxes and the potential linkage between this flux and

the H+ flux (Figure 4). Essential in this figure is the meaning of the graphs. By

convention a positive flux means an influx of the ions, both in the case of cations and

anions (Newman 2001). To relate this to the currents measured in electrophysiology

an influx of cations causes an inward current and a depolarization while an influx of

anions causes an outward current and a hyperpolarization.

Suddenly challenging the leaf tissue with 65/75 mM of NaCl caused the influx of

protons transiently to increase from around 25 μmol·m-2·s-1 to 150 μmol·m-2·s-1. After

20 minutes the proton influx returned to its pre-salinity values and started slowly to

change into a small efflux. Chloride fluxes were zero before NaCl was added but as

soon as 65 mM of Cl- was added this changed to an influx of chloride. However, no

differences in the response to the sudden application of NaCl of the mutants mutually

and wild type could be observed.

Figure 4. Typical changes in proton and chloride fluxes from wildtype Arabidopsis thaliana (upper panel) and the Atclca genotype (lower panel) leaf tissue induced by 65 mM of NaCl. The vertical line indicates the moment of the addition of 65 mM of NaCl. When the flux becomes more positive, this means either an increase of the influx or a reduction of the efflux. Typical experiments of at least three experiments are shown.


35

Phenotypical characterization of mutant plants

For none of the mutant lines a difference in their growth and development could

be observed when they were grown on soil (data not shown). The effects on

germination were studied on MS/2 agar media supplemented with different

concentrations (0-300 mM) of NaCl, KCl, KNO3 and cholinchloride (Figure 5). No

differences were observed between the six mutant lines mutually and in comparison

with wild type. However, germination was affected by the presence of different salt

species. Germination was the least inhibited by KCl. A 50% reduction of germination

was obtained at KCl concentrations above 275 mM. For both choline chloride and

NaCl the concentration at which 50% reduction of germination was obtained was

around 250 mM. The largest reduction in germination was observed when seeds were

allowed to germinate on KNO3, in this case 50% germination was already obtained at

200 mM.

In order to study the effect on growth and development plants were allowed to

germinate and grow on vertical plates under a large number of different physiological

conditions, like different salts, different concentrations of these salts and different pH-

values. Whereas, no differences could be observed when plants were grown under

standard conditions and the various salt conditions (data not shown), a difference in

the growth of some plant lines could be observed when the plants were grown on

MS/2 media with a higher pH: pH 6.2 instead of 5.8 (Figure 6). The single mutants

Atclca and Atclcd had a significantly reduced primary root length compared with the

wild type and the Atclcb mutant (Figure 6). Combining the mutations showed that

when AtClCb was also absent the effects of the absence of AtClCa and AtClCd were

reduced. The double mutant Atclcad showed no additive effect of the presence of both

disrupted genes. However, the triple mutant Atclcabd showed the largest reduction in

root elongation (Figure 6).

Chapter 2

36

Figure 5a. Germination of Arabidopsis seeds of the 8 genotypes on a half strenght MS medium, supplemented with the indicated salts. Upper panel: Concentration dependent inhibiton of germination. Lines are only drawn through the two outermost datasets. Datapoints are the average of 3 experiments and the error bars indicate the standard deviation. Lower pannel: Germination of the 8 genotypes at a single salt concentration. The data are same data as shown Figure 5a but for clarity shown separately. Datapoints are the average of 3 experiments and the error bars indicate the standard error


37

Figure 6 Upper panel: Primary root growth of the indicated Arabidopsis genotypes on a half strenght MS medium at two different pH values. Upper panel; pH 5.8; lower panel: pH 6.2. Length of the reference is 2.5 cm. Lower pannel: Length of the primary root of the indicated Arabidopsis genotypes on a half strenght MS medium at two different pH values. Datapoints are the average of 4 experiments and the error bars indicate the standard deviation. wt indicates wild type; a till adb indicate genotypes Atclca till Atlcabd.

Chapter 2

38

DISCUSSION

ClC proteins are an enigmatic group of proteins in plants. This family was named

based on the annotation of the first protein of this family, which was isolated from

Torpedo marmorata electric organ and characterized as being a chloride channel

(Jentsch et al., 1990). However, it has become oblivious that the function of these

proteins is not as clear cut as the name suggests. Reports have shown that these

proteins can function as channels but also can function as co-transporters and not only

as transporters of chloride but also of nitrate-ions (Bergsdorf et al., 2009).

The MIFE technique has shown to be very useful in assessing fluxes from and out

of almost intact tissues and the effect of various manipulations, which range from

elicitors and hormones to light and solutes (Lanfermeijer et al., 2008; Vreeburg et al.,

2005; Zepeda-Jazo et al., 2008). This technique is the link between electrophysiology

and whole plant physiology. We presently explored the potential of this technique to

study the physiological relevance of the Cl- fluxes in plants. Using a double barreled

configuration we assessed the changes of the H+ and Cl- fluxes when plants were

suddenly exposed to a concentration of 65/75 mM of NaCl. The addition of such a

concentration has two effects. Firstly, the external concentrations of Na+ and Cl-

change drastically and secondly the external water potential drops. As a result of the

latter water will move osmotically out of the cells resulting in a decrease of the turgor

pressure in the cell. Turgor pressure has been shown to control ion-fluxes, either via

mechano-sensitive channels (Chang et al., 1998) or via the plasma membrane H+-

ATPase (Sabala and Lew, 2002). We observed a transient increase in the proton efflux

upon NaCl exposure followed by a slow change into a small proton efflux. The time

course of this phenomenon can be compared with the observations of Shabala et al.

(2005) and Cuin et al. (2008), although those were made on roots. Their membrane

potential measurements show a transient depolarization and a partial recovery of the

membrane potential 10 to 15 minutes after the addition of salt. Our observed transient

influx of protons fits well with the transient depolarization and while the partial

recovery could be due to the emerging efflux of protons. However, Shabala et al.

(2005) did not observe this transient efflux of protons, they only observed the

decreasing influx. The increase of chloride influx is caused first of all by the

concentration gradient which favors an influx and, secondly, by the depolarization of

the membrane potential. Our observations are in accordance with those of Shabala et

al. (2005), who observed an increase of the chloride flux from 40 to 100 nmol·m-2·s-1


39

upon the exposing the barley leaf tissue to 20 mM of NaCl. The slow decrease of the

chloride flux could be ascribed to either the slow recovery of the membrane potential

or to the equilibration of the Cl- ions across the membrane (Figure 4).

We could not observe a difference between the response of the wild type and the

mutants in the MIFE system. Single mutants reacted in an identical manner to a

sudden exposure to 65 Mm NaCl (data not show), which implies that disruption of the

three ClC-proteins studied does not affect the observed fluxes.

Also no effects of the disruption of the ClC genes alone or in combination with the

others could be observed on germination. The effects of the different salt species

show that KCl is the preferred salt for germination. This salt probably is accumulated

rapidly and without any problems and increases the ability of the seedling to take up

water and grow. Sodium can not replace potassium in these experiments due its

toxicity and nitrate can not replace the chloride. Nitrate is probably partly assimilated

and turned into amino acids and proteins, while maybe at higher nitrate concentrations

the nitrate is not fully assimilates and becomes nitrite. Nitrite is toxic and will inhibit

growth. Hence, equivalent amounts of chloride and nitrate can not exert the same

effect on the water potential in growing cells.

Also other experiments failed to show differences between any of the mutants and

the wild type suggesting either delicate roles of the chloride channels, which could not

be observed by our crude observation methods. Moreover, it shows that the role of the

ClC proteins is not that obviously related with chloride and for instance salt and

salinity stress.

The experiments on root growth show an involvement of the ClC proteins with

growth. Two explanations can be given. Firstly, the AtClCa and AtClCd proteins are

involved in establishment of the proton-motive force (PMF). Root and cell growth, in

general, is driven by pH gradient across the cell. The proton motive force resulting

from this gradient across the membrane drives the uptake of solutes necessary from

generation of a low water potential, which subsequently is needed for the osmotically

driven uptake of water and the generation of turgor pressure. When taking the external

pH into account it is more difficult to establish the same PMF at the higher pH. This is

because at higher pH more protons are needed to achieve the same pH gradient. And

more protons transported across the membrane result in a larger membrane potential.

Hence, at higher external pH values it is advantageous to compensate for the positive

charge moved across the membrane when a proton is extruded in order to limit the

Chapter 2

40

polarization of the membrane. Polarization of the membrane impedes the transport of

the positively charged proton. Next to the coordinated influx of potassium ions the

coordinated efflux of chloride-ions can be used for this charge compensation. In this

model removing AtClCa and At ClCd hampers this process, reduces the generation of

the PMF and subsequently inhibits root growth.

Secondly, the AtClCa and AtClCd proteins are required for the accumulation of

the osmotically active anions, Cl- and NO3-, which is needed for cell expansion.

Absence of the transporters limits the uptake of these osmolytes and, thus, reduces

growth.

Recently a study appeared also describing a similar effect of the disruption of the

Atclcd mutation of root growth in relation with pH of the external medium (Fecht-

Bartenbach et al., 2007). They also suggested a role of the AtClCd protein in charge

compensation in relation with the establishment of a pH-gradient across the

membrane of a Golgi-derived transport vesicle. However, the AtClCd protein has all

characteristics of being an H+/2A- antiporter and this is difficult to match with a shunt

function of this system. Also AtClCa and AtClCb show characteristics which suggest

that these proteins are also H+/2A- antiporters, however, these proteins are considered

to be localized in the tonoplast membrane (Lv et al., 2009). The root-specific

expression of the three ClC proteins is highly similar, with expression mostly

restricted to maturation zone, and primarily in the vascular tissues (Lv et al., 2009)

although AtClCd seems to have considerable expression in the division zone as well.

In the study of Lv et al. (2009) the expression levels of AtClCa and AtClCd were

comparable, while AtClCb had a lower expression level. Although AtClCa and

AtClCd are localized in two different membranes their effect on root growth could be

similar. Cell growth needs an increase in volume but also an increase in surface or in

other words of plasma membrane area. The increase of volume requires the generation

of a PMF or the uptake of osmotically active solutes. If this is disturbed by disruption

of tonoplast-located AtClCa cells will not grow. The increase of plasma membrane

surface is accomplished by the continuous delivery of plasma membrane material

(lipids and proteins) by golgi-derived vesicles. The trafficking of these vesicles is

controlled by their internal pH. Hence, if the establishment of this pH is interrupted by

for instance the removal of AtClCd the plasma membrane can not increase its surface

area, hence, cells and, thus, roots stop growing.


41

Our results point to a role of ClC proteins closely related to energizing of the

membrane. Apparantly ClC proteins are important electrical circuits in the membrane

allowing the generation of steep ion gradients in combination with moderate

membrane potentials. As such, these proteins play an important role in the general

physiology of the cell. Further research will investigate the role and involvement of

ClC proteins in growth.

Chapter 2

42

ACKNOWLEDGEMENTS

I would like to thank Bert Venema for his help in the greenhouse.This work was

supported in part by a grant from the Ministry of Science, Research and Technology

of the Islamic Republic of Iran.

43

Chapter 3 NO3

- and H+ fluxes in Atclcd mutants of Arabidopsis thaliana


1 Department of Plant Physiology, University of Groningen, 9750 AA Haren, The Netherlands 2 Department of Agronomy and Plant breeding, Sari Agricultural Sciences and Natural Resources

University, Iran

Chapter 3

44

ABSTRACT

In the Arabidopsis genome seven ClC genes have been identified, including

AtClCa and AtClCd that are involved in nitrate accumulation in the vacuole and

cell expansion, respectively. The effect of NO3- on H+ and Cl- fluxes from leaf

tissue of Arabidopsis was determined for wildtype and and AtClCd T-DNA

insertion mutants. When leaf tissue of wildtype plants is exposed to increased

levels of nitrate in the external medium the influx of protons and chloride ions is

decreased. These effects are absent of much smaller in the AtClCd mutant plants

These results are consistent with a function of the AtClCd protein as H+/anion

antiporter.

NO3- and H+ fluxes in Atclcd mutants

45

INTRODUCTION

The protein family of chloride channels and anion transporters (ClC) is widely

distributed in prokaryotes and eukaryotes. They play important roles in cell signaling,

osmo-regulation, nutrient uptake and distribution, and metabolism. In higher plants

the first anion transporter proteins were described in tobacco and their identity was

inferred from homology with the ClC family of voltage-gated chloride channels in

animals (Lurin et al., 1996). Since then the function of only a few of these proteins in

plants has been established. In the Arabidopsis genome seven ClC genes can be

identified (AtClCa-g), and all of them have been isolated (Hechanberger et al., 1996;

Geelen et al., 2000; Lv et al., 2009). The intracellular localization of the respective

proteins was deduced from expression studies with green fluorescent protein (GFP)

fusion proteins (Hechenberger et al., 1996; Lv et al., 2009), but direct evidence for

their transport activity in plant cells is still lacking. GFP fusion protein studies point to

a subcellular localization of AtClCa, -b, -c and -g in the tonoplast, AtClCd and

AtClCf in the Golgi membrane and AtClCe in the thylakoid membrane (De Angeli et

al., 2007; Marmagne et al., 2007; Lv et al., 2009). However, most of these studies

used the 35S promotor, resulting in very high expression levels, possibly resulting in

a-typical localization of the fluorescent protein (see Chapter 1). Lv et al. (2009) found

that the highest expression of AtClCa and AtClCd is in the leaf and the root.

As uptake of Cl- is normally against its electrochemical gradient, which requires an

active mechanism, for instance, through a symporter with H+ as the second ion (the

suggested mechanism in plants (Felle, 1994)). Several mammalian ClCs have been

recognized to function as chloride/proton exchangers (Picollo and Pusch 2005; Scheel

et al., 2005).

In plants the function and mechanism of the different ClCs is still under debate. A

common characteristic of chloride channels is that they are also permeable for nitrate

(Pusch et al., 1995), a feature that in animals is of limited physiological importance.

In contrast, the nitrate transporter function of ClCs in plants could be the most

important one. In Arabidopsis thaliana AtClCa even appears to be much more

selective for NO3-, I- and Br- than for Cl- (De Angeli et al., 2006). As the

concentrations of cytosolic NO3- and Cl- in plant cells are approximately 4 and 10

mM, respectively (Felle, 1994; Miller and Smith, 1996; Lorenzen et al., 2004), plant

membrane potentials range from -150 to -220 mV (negative inside), and in most soils

the concentration of NO3- and Cl- is typically in the low millimolar to micromolar

Chapter 3

46

range (Marschner, 2002), the uptake of nitrate (and of chloride) is only possible by a

co-transporter system. Nitrate uptake in roots of Arabidopsis is mediated by the well-

characterized transporter proteins NTR1 and NTR2. The absorbed nitrate can either

be reduced by nitrate reductase, a cytosolic enzyme, or be transported to the shoot. In

contrast to the transporters that mediate the uptake into the root, the transporter

proteins that are involved in nitrate distribution in various tissues and in the nitrate

accumulation in the vacuole are less well studied. Mutant characterization studies

indicate that AtClCa (Geelen et al., 2000; De Angeli et al., 2006) and AtClCc (Harada

et al., 2004) are involved in the regulation of nitrate levels in Arabidopsis. Fecht-

Bartenbach et al. (2007) showed that AtClCd and V-ATPase support growth in

expanding cells and they suggest a more complex connections between ClC proteins

and the proton gradient. Disruption of AtClCd results in hypersensitivity to

concanamycin A, a specific inhibitor of V-type ATPase (Dettmer et al., 2006). De

Angeli et al., (2006) reported that AtClCa functions as a 2 NO3-/1 H+ antiporter that is

able to accumulate nitrate in vacuole. Lv et al., (2009), based on sequence comparison

with ClCs characterized in other species, postulated that AtClCd, which belongs to the

same subclass as AtClCa, may function as an anion/proton antiporter.

In the present study, the effect of nitrate in the experimental solution on Cl-, H+

and NO3- fluxes in leaf tissue of wildtype and Atclcd mutants was monitored with the

MIFE technique. The aim was to test the hypothesis that anion transporters located in

the tonoplast are essential in cytoplasmic pH homeostasis. Exposure to different

anions should not only lead to modifications in the fluxes of other anions, but also to

changes in the proton fluxes across the plasma membrane.


47


Plant material and growth conditions

Arabidopsis thaliana (wild type ecotype Columbia and the AtClCd T-DNA

insertional mutant) seeds were obtained from the Salk collection

(http://signal.Salk.edu/tdna_ protocols.html). For the AtClCd insertional mutant line

the SALK line 42895 was selected. Seeds were surface sterilized with gaseous

chlorine, sown in pots containing an organic-rich soil (TULIP PROFI No.4; BOGRO

B.V. Hardenberg, The Netherlands) and kept in the dark at 4oC for 3 days. The pots

were then transferred to a growth chamber with a 16h/8h light/dark cycle and a

temperature of 20±2oC for 20 days.

Selection and isolation of the T-DNA insertion mutant of Atclcd

The T-DNA insertion disrupting the AtClCd gene was identified in the database at

the SALK Institute Genome Analysis Laboratory. Homozygous mutants lines were

identified by screening for resistance to kanamycin and by a PCR-based screen with

selected primers for the gene and left border primer according to Salk protocol (see

Chapter 2). The plant line with the insertion in this gene is referred to as Atclcd.

Reverse Transcript PCR analysis

Total RNA was extracted from shoots and purified using the Qiagen RNeasy plant

mini kit according to the manufacturer’s protocol. RNA was measured by nano drop

machine and then first strand cDNAs was synthesized from total RNA (2μg) isolated

using reverse transcriptase (Fermentas, USA) and oligo (dT) primer. Tubulin primers

were included, for presence of equal amount of cDNA. For amplification, PCR was

performed at an annealing temperature of 55oC, using 32 cycles.

MIFE measurements

Net fluxes of Cl-, NO3- and H+ were measured non-invasively using the MIFE

(Micro Electrode Flux Estimation) technique essentially as described in Shabala et al.,

(1997), Newman (2001) and Lanfermeijer et al. (2008). Briefly, microelectrodes were

pulled and then dried in an oven at 200oC overnight. To improve the stability of the

liquid ion exchange cocktails (LIX) the electrodes were coated with a hydrophobic

material (tributylchlorosilane 90796; Sigma-Aldrich, Milwaukee) for 10 min in the

Chapter 3

48

same oven under a steel cover. Then the cover was removed and the electrodes were

left to dry at 200oC for another 20 min.

The electrodes were back-filled with 0.5 M KCl in the case of Cl--specific

electrodes, 15 mM NaCl + 40 mM KH2PO4 in the case of H+-specific electrodes and

0.5 M KNO3 + 0.1 M KCl in the case of NO3--specific electrodes. All the back-fill

solutions were adjusted to pH 6 with NaOH. Immediately after back-filling, the

electrode tips were front-filled either with a commercially available LIX, ionophore

24902 for Cl- (Cl- -specific electrodes were used after an overnight ‘maturation’

period) and 95297 for H+ (Fluka; Busch, Switzerland) or with a LIX consisting of

0.5% methyltridodecylammonium nitrate (MTDDA), 0.084% methyl-triphenyl-

phosphonium bromide (MTPPB) and 99.4% n-phenyloctyl ether (NPOE) for the NO3-

-specific electrodes (Table 1).

Ion-specific Electrode

Electrode tip fill Backfill Specificitya Responseb (mV/decade)

H+ Ionophore 95297 15 mM NaCl,

40 mM KH2 PO4

H+/Li+=108 51

Cl- Ionophore 24902 0.5 M KCl Cl-/I-=10 48

NO3- 0.5% MTDDA NO3

-c,0.084% MTPPBd and 99.4% NPOEe

0.5 M KNO3, 0.1 M KCl

Cl-/I-=8 56

Table 1: Composition of the ion-specific electrodes and their characteristics as used in the MIFE.experiments. a: specificity ration with the ion which interferes the most with the studied ion. b:The response value is the change in the measured potential when the pIon (e.g. pH, pCl or pNO3) changes 1 unit.c: methyltridodecylammonium nitrate; d: methyl-triphenyl-phosphonibromide; e: n-phenyloctyl ether

The epidermis was removed from the abaxial side of the leaf. Leaf material was

immobilized on a glass capillary using grease with the abaxial side exposed to the

solution and was placed in a measuring chamber with a transparent bottom. The

chamber was filled with 1 ml of the basic measuring solution (1 mM KCl, 0.5 mM

CaCl2, pH 5.8 for H+ and Cl- measurements or 0.1 mM NH4NO3, 0.2 mM CaSO4, pH

5.8 for NO3- measurements), submerging the leaf material. The whole chamber was

placed on a Nikon TMS inverted microscope. The ion-selective microelectrodes were

mounted at an angle between 30o and 40o with the horizontal in a holder (MMT-


49

5Narishige) on a micromanipulator (PCT; Luigs and Neumann) that was driven by a

computer-controlled motor (MO61-CE08; superior Electric). All electrodes were

calibrated before and after use in a series of solutions with concentrations in the

expected range of the ions in the experimental solutions. The medium in the chamber

was continuously replaced using a flow-through system (with a flow rate of

approximately 3 ml/min). A system of taps allowed changes of the medium from

outside the Faraday cage, which enclosed the whole set-up. Net fluxes of Cl-, NO3-

and H+ were recorded in response to exposure of the leaf material to solutions of

different solute composition (Table 2).

Measurement Conditions applied

NaCl CholinCl Mannitol KNO3 KCl

H+ Fluxes + + + + +

Cl- Fluxes + + + +

NO3- Fluxes + +

Table 2. Osmotic conditions tested on Arabidopsis wild type and mutant plants for the indicated flux measurements. “+” indicated monitored

Chapter 3

50

RESULTS

Isolation of a homozygous knockout line and gene expression

We obtained one T-DNA insertion line in the Colombia ecotype background from

the Salk collection under number SALK-42895. The exact location of the T-DNA

insertion for the locus At5g26240 was obtained from the TAIR database

(http://www.arabidopsis.org). As shown in figure 1, the T-DNA insertion in Salk-

42895 was located in the fourth intron of the gene. The location of the T-DNA in the

gene was confirmed by PCR according to the protocol of the SALK consortium. To

study AtClCd gene expression, RT-PCR was performed using gene specific primers.

Figure 1 shows the expression of AtClCd, using tubulin as an internal standard. The

RT-PCR products confirmed that high expression levels of AtClCd are present in the

shoot. RT-PCR also confirmed that in homozygous mutant plants the full transcript of

AtClCd is absent (Figure 1). This suggests that the T-DNA insertion results in a null

allele. Our results confirm an earlier study that showed high expression levels of

AtClCd in root and shoot (Lv et al., 2009).

Figure 1. The T-DNA insertion in the AtClCd gene a: A schematic representation of the position of the T-DNA insertion in AtClCd gene. b: Tubulin primer experiment showing presence of equal amount of cDNA. c: The absence of expression in AtClCd gene in its T-DNA insertion lines. The wildtype and the Atclcd genotype were analysed using the primers shown in table 1 of Chapter 1. The Tubulin transcript levels are shown as a loading control.

KNO3–induced proton fluxes are different in wildtype and Atclcd mutant plants

Based on the similarity of AtClCa and AtClCd we hypothesize that AtClCd is also

located in the tonoplast, functions as a H+/NO3- antiporter and is involved in nitrate

accumulation in the vacuole. Based on these characteristics we predicted that

increasing the extracellular nitrate concentration would lead to distinct differences

between the Atclcd mutants and wildtype plants: 1. Since in the mutant excess nitrate


51

cannot be stored efficiently in the vacuole, the cytosolic pH will increase more in the

mutants compared to wildtype. In wildtype plants the cytosolic pH will be kept lower

due to the exchange of nitrate from the cytosol for protons from the vacuole.

Furthermore, the reduction of nitrate in the cytosol will consume one proton per NO3-

reduced to nitrite and several more when reduced further to ammonia. 2. The influx of

nitrate will affect the chloride influx in the mutants more than in wildtype plants as

accumulation of nitrate in the cytoplasm will likely reduce further uptake of anions. 3.

The increase in the nitrate influx is expected to be transient in both genotypes, but will

be more pronounced in wildtype plants since the capacity to store nitrate is higher.

Addition of nitrate (400 µM and 6 mM KNO3) resulted in an immediate increase in

the influx of nitrate in the leaf tissue (Figure 2). The size of the transient increase of

the nitrate influx did not differ significantly between wild type and AtClCd mutant

plants.

Increasing the nitrate concentration in the external medium resulted in a reduction

of the proton influx in wildtype plants (Figure 3). In Atclcd mutant plants nitrate

increased the proton influx even further. When DCCD was added to the medium the

influx of protons was dramatically reduced, while in wildtype plants the influx

remained at the same low level.

Effect of external KNO3 on chloride flux

In order to check the effects of increasing the external concentration of nitrate on

chloride fluxes, we measured Cl- fluxes before and after nitrate treatment. AtClCa has

been shown to be selective for both NO3- and Cl-. Furthermore, NO3

- generally

suppresses Cl- fluxes and accumulation (Bar et al., 1997; Kafkafi et al., 1982; Adler

and Wilcox, 1995) and decreases the Cl- influx, in particular the flux into the vacuole

(Britto et al., 2004). As shown in figure 4 250 μM KNO3 indeed decreased the

chloride influx and induced an efflux. In contrast, in Atclcd mutant plants, addition of

KNO3 only has a transient effect on chloride flux and concentration, as after

approximately 1.5 minute the flux returns to pre-addition values.

Chapter 3

52

Figure 2. Typical changes in nitrate fluxes from wildtype Arabidopsis thaliana (panels A and B) and the Atclcd genotype (panels C and D) leaf tissue induced by 6 mM (panels A and C) or 400 μM of KNO3 (panels B and D). The vertical line indicates the moment when the basal salt medium was slowly replaced by BSM with the supplement. When the flux becomes more positive, this means either an increase of the influx or a reduction of the efflux. Typical experiments of at least three experiments are shown.

Figure 3. The effect of DCCD on nitrate induced proton fluxes from wildtype Arabidopsis thaliana and the Atclcd genotype leaf . The vertical lines indicate the moments when the medium was slowly replaced by the next medium as indicated. The concentations used were 50 mM KNO3 and 20 µM DCCD. Typical experiments of at least three experiments are shown.


53

DISCUSSION

Relation between NO3- and H+ fluxes in Atclcd mutant plants

The physiological characterization of Arabidopsis mutants suggested the

involvement of AtClCa (Geelen et al., 2000; De Angeli et al., 2006) and AtClCc

(Harada et al., 2004) in the regulation of nitrate levels in plants. The bacterial ClC-ec1

protein (Accardi and Miller, 2004) and the human ClC4 and ClC5 proteins (Picollo

and Push, 2005; Scheel et al., 2005) that are located in the membranes of intracellular

vesicles, have been shown to function as proton/chloride exchangers, rather than

passive chloride channels. In plant cells AtClCa functions as a 2 NO3-/1 H+ antiporter

facilitating the accumulation of nitrate in the vacuole (De Angeli et al., 2006), with a

selectivity sequence of NO3- = I- > Cl-. AtClCd has been shown to co-localize in the

trans-Golgi network with VHA-a1, a subunit of the proton transporting V-Type

ATPase (Fecht-Bartenbach et al., 2007).

In plant cells, the plasma membrane H+-pumping ATPase is the primary active

transport system and responsible for generating the membrane potential (Assman and

Haubrick, 1996). Transport of a cation in the opposite direction or an anion in the

same direction is required prevent extreme hyper-polarization of the membrane and

allow the build up a steep proton gradient. Therefore different factors like activation

of anion channels or changes in pH of the medium may lead to changes the membrane

potential and the cytosolic pH. Also plasma membrane anion channels play a central

role in the regulation of the cytosolic pH of plant cells (Johannes et al., 1998). The

results of our experiments show that application of KNO3 leads to an H+ efflux (seen

as a reduction of the influx). This efflux is, as is evident from its sensitivity to the

ATPase inhibitor DCCD, carried by the plasma membrane H+-ATPase. This NO3--

induced H+ efflux confirms reports by Garnet et al. (2003) on eucalypt and Segonzac

et al. (2007) on Arabidopsis. In mutant plants the addition of KNO3 has much less of

an impact on the H+ fluxes, demonstrating that the presence of an H+/anion antiporter

is essential for the nitrate-induced effects on the plasma membrane proton pump.

Interactions between uptake of Cl- and NO3- ions in Atclcd mutant plants

Under natural condition, the presence of nitrate in the soil can reduce the toxic

effect of excess Cl- (Bar et al.,1997). Nitrate can reduce the influx Cl- in plant cells

Chapter 3

54

and Cl- accumulation in plant tissue (Glass and Siddiqi, 1985; Adler and Wilcox,

1995). In our experiments the addition of NO3- led to an increase in the Cl- efflux

(decreased Cl- influx) and to a higher external Cl- concentration when wildtype plants

were studied. In the Atclcd mutant plants addition of nitrate resulted in a short,

transient change in the chloride flux, but not in a sustained efflux (Figure 4). These

results are consistent with a function for AtClCd as an H+/anion antiporter that can

transport both nitrate and chloride across the tonoplast.

Figure 4. Typical changes in chloride fluxes from wildtype Arabidopsis thaliana and the Atclcd genotype leaf tissue induced by 250 μM KNO3. The vertical line indicates the moment when the basal salt medium was slowly replaced by BSM with the supplement. Typical experiments of at least three experiments are shown.

Although the selectivity of the bacterial and the Arabidopsis ClC transporters is

clearly different, (in Arabidopsis NO3-, I- and Br- > Cl- (De Angeli et al., 2006), in

bacteria Cl- > Br-, NO3- and SO4

- (Accardi and Miller, 2004)), it is clear that in both

NO3- and Cl- could enter the cells via the ClC transporters. Furthermore, addition of

NO3- decreases the Cl- influx into the vacuole (Britto et al., 2004). These and our data

suggest that in plant cells the accumulation of chloride and nitrate in the vacuole is

based on competition for transporter activity and that the transporter that mediates this

transport might be the AtClCd protein.


55

ACKNOWLEDGEMENTS

We thank Marten Staal for excellent technical assistance with the MIFE

technique. This work was partially funded by a grant from the Ministry of Science,

Research and Technology of Islamic Republic of Iran.

57

Chapter 4 Anion Channels and Root Elongation in Arabidopsis thaliana


1 Department of Plant Biology, University of Groningen, 9750 AA Haren, The Netherlands 2 Department of Agronomy and Plant breeding, Sari Agricultural Sciences and Natural Resources

University, Iran

Chapter 4

58

ABSTRACT Anion transporting proteins belonging to the chloride channel (ClC) family

are involved in anion homeostasis in a variety of organisms. Progress in the

understanding of their biological functions is limited by the small number of

genes identified so far. Seven chloride channel members could be identified in

the Arabidopsis genome, amongst which AtClCa, AtClCb, and AtClCd are more

closely related to each other than to the other plant ClCs in same subclass.

Chloride channels from Arabidopsis have been shown to participate in nitrate

accumulation and storage. In this study, the physiological role of AtClCa,

AtClCb and AtClCd proteins was investigated. Disruption of the AtClCa,

AtClCb and AtClCd gene by a T-DNA insertion did not yield a phenotype that

was different from wildtype under normal conditions, however, when the pH of

the medium was slightly less acidic (raised from 5.8 to 6.2) the length of the

primary root of plants with a disrupted AtClCa and AtClCd gene was reduced

compared to wildtype and the plant with a disrupted AtClCb gene.

The proton fluxes and pH were measured along the surface of the root at

different positions, from root cap, through the transition zone, and up to the fast

elongation zone, and at different pH’s of the medium. A high proton influx was

found in the apical part of the transition zone. Lower influxes or even small

effluxes were found at the basal part of the elongation zone. At pH 6.2 the influx

of protons in the apical part of the transition zone in the Atclca and Atclcd

mutants was significantly lower than in wildtype and the Atclcb mutant.

Measurement of the distance between root tip and first epidermal cell with

visible root hair bulge indicate that the mutants that are affected in the H+ flux,

the Atclca and Atclcd mutants, also have a reduced cell expansion. A model for

the interaction between endomembrane anion/H+ antiporters, plasma membrane

proton fluxes and cell expansion is discussed.

Anion channels and root elongation

59

INTRODUCTION

Root development is determined by cell division, differentiation and expansion.

Each of these processes is under the control of an intrinsic developmental program

and of external biotic and abiotic factors (Lynch, 1995). A wealth of information is

available on the cellular organization, the differentiation and genetic background of

developmental processes in the root of Arabidopsis, the model system of choice for

plants (Dolan et al. 1993; Schiefelbein et al., 1997; Scheres et al., 2002; Park et al.,

2008). Several groups have explored the mechanisms of cell expansion in the

elongation zone, the zone of maximal cell growth rate (Befey et al., 1993; Hauser et

al., 1995; Verbelen et al., 2001; Swarup et al., 2007; Cnodder et al., 2006). Cell

elongation in roots is sensitive to various endogenous and exogenous factors such as

pH (Rayle and Cleland, 1970), ethylene (Le et al., 2001), auxin (Fujita and Syono,

1996), calcium (Kiegle et al., 2000) and aluminium (Sivaguru et al., 2000). The

primary cell wall of plants consists of long cellulose microfibrils embedded in a cross-

linked matrix of polysacharides, largely pectin and glycans (Carpita and Gibeaut,

1993), and a small quantity of structural proteins (Showalter, 1993). The acid growth

theory states that protons are the primary wall-loosening factor, causing the cleavage

of load-bearing bonds in the cell (Rayle and Cleland, 1970; Royle and Cleland, 1992).

Turgor will then cause a cell with a loosened cell wall to expand. In maize, the spatial

profile of growth along the roots has been shown to coincide with the spatial profile of

root-surface acidification (Fan and Neuman, 2004; Peters and Felle, 1999; Pilet et al.,

1983). A more recent version of the acid-growth theory states that a low apoplastic pH

(<5) activates expansins, cell wall-assiociated proteins that break the hydrogen bonds

between the cellulose chains and the cross-linking glycans (Mc Queen-Mason et al.,

1992; Cosgrove, 2000). The apoplastic pH is determined by the H+-efflux through the

plasma membrane H+-ATPase and the H+-influx through the H+-coupled anion

symporters (Taner and Caspari, 1996). Anion fluxes through anion channels may

contribute to the maintenance and regulation of proton gradients across the different

membrane compartments in plant cell. Based on transport studies and structure-

function relationships the AtClCa and AtClCd proteins are very likely to function as

anion/proton antiporters (De Angeli et al., 2007; Lv et al., 2009). In combination

AtClCd and V-ATPase can support expansion growth of cells. This last result

suggests more complex connections between ClC proteins and proton gradients

(Angeli et al., 2006; Jennifer et al., 2007 and Scheel et al., 2005). As shown by GUS

Chapter 4

60

staining, the elongation and maturation zone of the root show high expression levels

of AtClCa and AtClCd (Lv et al., 2009). Although there are enough indications that

ClC proteins are essential in cell expansion in certain tissues and cell types, the

functional relation between AtClC proteins and proton gradient development in

expanding root cells is still unclear. In this study, we tried to elucidate the relationship

between ClC transporter proteins and the proton flux in root cell elongation, by

quantifying the fluxes along the root in wildtype and the Atclca, Atclcb and Atclcd

mutants at different external pH’s.


61


Plant materials and culture conditions

The seeds of Arabidopsis thaliana (ecotype Columbia) were obtained from the

SALK collection (AtClCb: SALK27349; AtClCd: SALK42895) and from the

WiscDsLox T-DNA collection (AtClCa: WiscDsLox477-480I4). Seeds were surface

sterilized with gaseous chlorine and sown in 90 mm petridishes containing with half-

strength Murashige and Skoog media (Duchefa, Haarlem, The Netherlands) with

0.8% w/v micro agar (Duchefa, Haarlem, The Netherlands). The dishes were sealed

with surgical tape and incubated in the dark at 4 0C for 3 days. Subsequently they

were transferred to a growth chamber (set at a 16h/8h light/dark cycle, 20 ± 20C

temperature at 72% relative humidity) and placed on edge, 5 degrees off the vertical,

such that the roots were growing down along the surface of the agar without

penetrating it. About 4-6 days and 14 days old plants were used for MIFE and primary

root measurement, respectively.

Ion flux experiments

Net fluxes of protons were measured non-invasively using vibrating H+-selective

microelectrodes with the MIFE (microelectrode ion flux estimation) technique

(Shabala et al., 1997 ; Newman, 2001; Vreeburg et al., 2005; Lanfermeijer et al.,

2008). Micropipettes (diameter 50 μm) were pulled from borosilicate glass. The

electrodes were silanized with tributylchlorosilane (Fluka 90974) and subsequently

back-filled with 15 mM NaCl and 40 mM KH2PO4 and front filled with Hydrogen

Ionophore II, Cocktail A (Fluka 95297). Only the electrodes with a response between

50 and 59 mV per pH unit and with a correlation coefficient between 0.999 and 1.000

(pH rang 5.1-7.8) were used. The electrodes were calibrated before and after use.

Roots of five days old Arabidopsis seedlings were mounted on glass capillary tubes

with medical adhesive and placed in a measuring chamber with a transparent bottom,

which was filled with BMS solution (1 mM KCl, 0.5 mM CaCl2, pH 5.8 for H+ and

Cl- measurements). The whole chamber was placed on the stage of a Nikon TMS

inverted microscope.

The H+-microelectrode was mounted at an angle between 300 and 400 with the

horizontal in a holder (MMT-5; Narishige) on a micromanipulator (PCT; Luigs and

Neuman) driven by a computer-controlled motor (MO61-CE08). The electrode was

Chapter 4

62

positioned manually at a distance of 10 μm from the root. During the subsequent

measurement, the distance between the electrode and the surface of the root was

switched between 10 μm and 50 μm at a frequency of 0.1 Hz. The chemical activity of

H+ in solution at these two positions was recorded and from these data the H+-flux and

the pH could be calculated.

The absolute pH value could differ (± 0.1-1 pH units) between different MIFE

experiments, but the overall pattern of the pH along the root stayed the same. The first

measuring point was positioned at a root tip and the subsequent sampling points along

the root were 75 μm apart. At each measuring point the ion flux was recorded for 2 min.

The last sampling point was chosen at the beginning of the root hair zone.


Homozygous mutants lines were identified by resistance to kanamycin and by a

PCR-based screen with the left border primer (LB) according to the Salk protocol and

the respective primers (has been described in chapter 2)

Expression analysis in roots

Total RNA were isolated from roots using a Nucleospin RNA plant kit

(Macherey-Nagel). RNA was measured by the nanodrop machine. Total RNA (3μg)

were used as template for first-strand cDNA synthesis using 200U of RevertAid H-

Minus M-MuLV reverse transcriptase (Fermentas, www.fermentas.com) and an

Oligo (dT) primer. As a control for equal amounts of cDNA tubulin primers were

included (Figure 1). PCR was performed at an annealing temperature of 55 oC and 32

cycles were used for AtClCa and AtClCd and 35 cycles were used for for AtClCb.

Primers are given in table1 chapter 2.

Figure 1. The absence of expression of the AtClCa, AtClCb and AtClCd genes in their respective T-DNA insertion lines. The four genotypes were analysed using the primers shown in table 1 of chapter 2. upper panel: the Tubulin transcript levels of the four genotypes are shown as a loading control. MW: lane with the molecular marker, the size of the essential bands is shown on the left. lower panels: expression levels of the AtClCa (left panel), AtClCb, (middle panel) and AtClCd (right panel) in wildtype and the respective T-DNA insertion lines.


63

Cell imaging in primary root

Images were taken with a Nikon Coolpix 990 digital camera, which was mounted

on an inverted optical microscope (CX41, Olympus, Tokyo, Japan) equipped with

objectives of 20× and 40× magnification. After 7 and 14 days of growth on the

vertical-placed plate the distance between the root tip and the first epidermal cell with

visible root hair bulge (DFEH) and the root length were measured. At least 10

Arabidopsis wildtype and mutant plants were measured for every condition in each

experiment and each experiment was repeated 3 times.

Chapter 4

64

RESULTS

Isolation of homozygous knockout lines

From the seven members of the ClC transporter protein family in Arabidopsis

thaliana tree genes (AtClCa, b and d) that are phylogenetically more closely related to

each other than to any of the other members were selected. Plant ClC proteins are

grouped into two distinct subclasses with significant divergence (Lv et al., 2009). In

subclass 1 AtClCa andAtClCb are the most closely related, while AtClCd although in

another branch of the same subclass, is also rather similar. Of the other proteins in this

family, AtClCc and AtClCg, also belong to subclass 1, while AtClCe and atClCf,

belong to subclass 2 and are more distantly related. Homozygous T-DNA insertion

lines in the Columbia ecotype were selected from Salk and the WiscDsLox T-DNA

collections.

Reverse transcript PCR analysis of gene expression

As shown in figure 1, RT-PCR confirmed high expression levels of AtClCa,

AtClCb and AtClCd in root tissue of wild type plants. This result is in agreement with

an earlier study (Lv et al. 2009) with showed high expression in the root of AtClCa

and AtClCd and moderate expression of AtClCb. In the homozygous mutant plants

transcripts of the respective disrupted genes could not be detected (Figure 1).

Primary root growth of Atclca and Atclcd inhibition at high pH

Since anion transporters are involved in osmo-regulation and in cell expansion, we

measured the primary root length of wildtype and mutant plants growing on agar

plates and exposed to different external pH’s (5.8 till 6.8, buffered with 20 mM Mes).

All genotypes showed the longest root when grown on media with a pH of 5.8, while

no differences could be observed between the genotypes (Figure 2). When the pH of

the medium was raised root growth decreased (Figure 2), however, compared to the

wildtype, root growth in Atclca and Atclcd was more reduced. Atclcb was not

distinguishable from wildtype at all pH values. In all 8-days-old plants exposed to the

highest pH (6.8) the roots of the seedlings had hardly grown at all and the leaves were

yellowing.


65

Reduced proton flux on the growth zones of root Atclca and Atclcd

To detemine if the clear difference in root length between wildtype plants and

Atclca and Atclcd mutants at less acidic pH’s, could be correlated to differences in cell

wall acidification, the proton fluxes at the surface of the roots of 8-days-old plants

were measured in wildtype and mutant plants in media with different pH’s. The pH

and H+ flux profile along the root was recorded at 75 μm intervals. The last sampling

point was

Figure 2. The effect of the pH of the growth medium on primary root length of wildtype Arabidopsis and the three single mutant lines. a: Phenotypes of roots of the wildtype and the three single mutants grown at different pH-values. The distance between two short lines on the reference is 1 mm. b: Quantification of primary root length of wildtype and the three single mutants grown at different pH-values. Datapoints are the average of 4 experiments and the error bars indicate the standard deviation.

chosen at the onset of the root hair zone. At pH 5.8 the largest influxes were recorded

at a distance of 225 to 250 μm from the root tip, which is the border between the

meristematic zone (MZ) and the transition zone (TZ) (Figure 3). In the transition zone

the influx decreases steeply and remains relatively stable throughout the elongation

zone. At pH 5.8 no differences between wildtype and mutants can be observed

(Figures 3 and 4).

Chapter 4

66

At higher pH’s (in figures 3 and 4 the results for pH 6.2 are shown) the largest net H+

influx is shifted slightly basipetally to 300 μm from the root tip in wildtype and

Atclcb. However, more significant was the almost complete disappearance of the H+

influx in the Atclca and Atclcd mutants.


67

Figure 3. The proton flux profile along the roots of wildtype and the three single mutants. Upper panel: proton fluxes measured at pH 5.8, lower panel: proton fluxes measured at pH 6.2. Indicated are the three zones of the growing roottip: the meristematic zone (MZ), the transition zone (TZ) and the elongation zone (EZ). Datapoints are the average of 4 experiments and the error bars indicate the standard deviation

Difference DFEH between wildtype and Atclc mutant plants

The acid growth theory predicts cell wall loosening and rapid cell elongation at

low pH. In order to check the relation between different pH’s and cell elongation in

the primary root, we measured the distance between root tip and the first epidermal

cell with visible root hair bulge (DFEH). In 8-day-old plants grown at pH 5.8 DFEH

was 1355 ± 15 μm (Figures 4 and 5). At pH 6.2 DFEH was decrease in all plants, but

significantly more so in the Atclca and Atclcd mutants.

Chapter 4

68

DISCUSSION

Expansion of cells is only possible when the yield threshold of the cell wall is low

enough and the turgor, the pressure the cell exerts on the cell wall, high enough. For

both of these parameters a close interaction between transporter proteins is necessary.

The apoplastic pH of root cells will reflect the pH of the medium, but is also

determined by the H+-efflux mediated by the plasma membrane H+-ATPases and the

H+-influx through the H+-coupled anion symporters (Tanner and Caspari, 1996). The

plasmamembrane proton pumping ATPase activity is also one of the main regulators

of the cytoplasmic pH stat. An increase in cytoplasmic pH will necessarily result in

down-regulation of the H+-ATPase activity. Any transport process, also across

endomembranes, that affects the cytoplasmic pH is likely to affect the net proton

fluxes at the cell surface. Hence, the presence or absence of an anion/H+ antiporter

will have such an effect.

Figure 4. Comparison of the peak values of the proton-flux (upper panel) and the DFEH (lower panel) at two pH values. Upper panel: peak values are the fluxes measured around 250 μM from the roottip as shown in Figure 3. Lower panel: DFEH: the distance between root tip and first epidermal cell with visible root hair bulge. Datapoints are the average of 6 experiments and the error bars indicate the standard deviation.

For the second requirement for cell expansion, the generation of sufficient turgor,

the same anion/H+ antiporter will also have a key role. Accumulation of solutes that

have to provide the low osmotic potential to attract water to enter the cell will have to

be balanced in all cellular compartments. Therefore, the vacuole, being the largest

compartment has to be stocked with a mixture of small organic molecules and nearly


69

equal amounts of cations and anions. Under most conditions the accumulation factor

between cytoplasm and vacuole found for anions can thermodynamically not be

explained by simple diffusion down the electrical potential (positive inside the

vacuole). The accumulation of anions is often so high that secondary active transport,

mediated by anion/H+ antiporters is essential. Also plasma membrane anion channels

play a central role in cytosolic pH regulation of plant cells (Johannes et al., 1998).

The link between ClC anion transporters and H+-pumping has been confirmed for the

mammalian ClC3, ClC5 and ClC7 transporters (Jentsch et al., 2002). The prokaryotic

ClC anion channel was shown to mediate a stoichiometrically fixed 2 anions/proton

antiport activity (reviewed in Miller, 2006). In plants, the same function has been

proposed for AtClCa and AtClCd (De Angeli et al., 2006 and De Angeli et al., 2007).

After confirmation that the Atclca, -b and -d mutants we had selected for this study

were homozygous, and indeed lacked a full transcript (Figure 1) we used them to

elucidate the role of endomembrane H+/anion antiporters in Arabidopsis root

elongation.

Proton fluxes and root cell elongation

Changing the pH from 5.8 tot 6.2 reveals three differences between Atclca and

Atclcd on the one hand and wildtype and Atclcb on the other. In Atclca and Atclcd

increasing the pH leads to 1) a more drastic decrease in H+ influx, 2) a stronger

inhibition of primary root growth and 3) to a shortening of the root expansion zone.

From these results we conclude that the AtClCa and AtClCd transporter proteins

are involved in primary root expansion growth. This conclusion is based on the

following considerations: 1) For the different genotypes exposed to a higher pH, the

length of the primary root correlates with the distance between root tip and first

epidermal cell with visible root hair bulge (DFEH) and this indicates that specifically

cell expansion is reduced in the Atclca and Atclcb mutant plants. The length of the

first epidermal cell with a visible root hair bulge (LEH) was previously defined as a

parameter to study root development and the control of elongation on cell level (Le et

al., 2001). This parameter is less useful when cell size measurements are more

difficult to perform, which for instance is the case when the root tips are swollen and

have accumulated pigments, and the epidermal cell walls are obscured. Measuring the

DFEH is easier since it only involves the recognition of the first root hair bulge and

the root tip. Since the epidermal cell exhibiting the first root hair marks the end of the

Chapter 4

70

fast elongation zone and the onet of the differentiation zone in Arabidopsis root (De

Cnodder et al., 2006) the DFEH should therefore give a fairly accurate reflection of

the expansion rate in the distal part of the primary root. This implies that in Atclca and

Atclcb mutants the expansion rate is reduced compared with wildtype and Atclcb

(Figures 4 and 5).

Figure 5. Phenotypes of the roots of the Arabidopsis wildtype and the three single mutants when grown on media with different pH-values. The first epidermal cell with a visible root hair bulge is indicated by a arrow. This cell is used to measure the DFEH (distance between root tip and first Epidermal cell with visible root hair bulge). The bar in the upper left photo indicates 0.5 millimeter.

2) The zone of highest expression of the ClC genes, that show a reduced elongation

rate when mutated, coincides with the elongation zone. By using GUS staining in the

root Lv et al (2009) showed that AtClCa and AtClCd have the highest expression in

the elongation and maturation zone, but that they are absent in the division zone.

3) A function of AtClCd in cell expansion in root growth has been proposed

earlier. Atclcd mutants plant exhibit a reduction in root growth when compared to

wildtype at elevated pH’s of the medium, which is also attributed to low cell

expansion rates (Fecht-Bartenbach et al. 2007). The AtClCd protein is essential for

normal cell expansion of hypocotyls cells in which the V-type ATPase is inhibited or

only partly functional (Fecht-Bartenbach et al., 2007).

The result that the mutants with a more strongly reduced H+ influx, are the most

severely inhibited in root growth, is not immediately consistent with the accepted acid

growth theory for expansion growth. Normally, reduction of H+ influx would result in


71

a lowering of the apoplastic pH and, consequently, it would be expected that the

expansion growth is stimulated in this situation. In the literature the indications that

lowering the pH induces cell elongation are many (Taguchi et al., 1999; Vanderhoef

and Dute, 1981; Rayle and Cleland 1992). For instance, part of elongation zone

growth is regulated by acid growth phenomena associated with cellular control over

the cell wall pH (Edwards and Scott, 1974; Buntemeyer et al., 1998; Peters and felle,

1999). Under normal conditions the surface pH along Arabidopsis roots, is highest in

the transition zone, and lowest in the adjacent fast elongation zone (De Cnodder et al.,

2006). Our results thus do not fit with this general model. We find that faster

elongation correlates with higher influx, not with higher efflux. We hypothesize that

for maintaining root growth at higher pH, functional AtClCa and AtClCd proteins are

necessary to drive the accumulation of anions in intracellular compartments,

specifically the vacuole and/or acidic vesicles, and to generate sufficient turgor. This

hypothesis would fit with our results: 1) the increased proton efflux in wildtype is

possibly the result of sustained anion/H+ co-transporter activity in the plasma

membrane and 2) the reduced root length phenotype of the mutants is only obvious at

higher pH values. At these higher pH’s cell wall elasticity is lower and elongation will

only be possible by higher turgor values.

Chapter 4

72

ACKNOWLEDGEMENTS We would like to thank Ger Telkamp for his help in culturing the plants and

Marten Staal for his expert support with the MIFE measurements. This work was

supported in part by a grant from the Ministry of Science, Research and Technology

of the Islamic Republic of Iran.

73

Chapter 5 The role of AtClCa and AtClCd in heavy metal tolerance in

Arabidopsis thaliana


1 Department of Plant Biology, University of Groningen, 9750 AA Haren, The Netherlands 2 Department of Agronomy and Plant Breeding, Sari Agricultural Sciences and Natural Resources

University, Iran

Chapter 5

ABSTRACT

In higher plant cells, anion channels play a role in acclimation of plant cells

to abiotic and biotic environmental stresses, in the control of metabolism and in

the maintenance of electrochemical gradient. A number of studies have

demonstrated that anion channels are present in various cell types of plants.

Seven genes encoding chloride channel (ClC) proteins have been identified in the

Arabidopsis genome. To assess the function of ClCs in Arabidopsis, we obtained

three single mutant plants (Atclca, Atclcb, and Atclcd) with an T-DNA in the

respective genes and we made the three double and the triple mutant plants.

One of the aims of our study was to elucidate the role of anion channels in heavy

metal (i.e. cadmium) resistance and specifically their involvement in the

alleviation of cadmium effects by calcium.

The primary root growth and the morphology of cells between the

meristematic and elongation zone, were significantly affected by exposure to 60

μM Cd+2 in all genotypes. Adding Ca2+ to Cd2+-exposed plants, restored primary

root growth and the normal shape of cells, except in the double mutant Atclcad

and the triple mutant Atclcabd. We propose that both AtClCa and AtClCd

proteins play a role in the detoxification of cadmium by allowing efficient

sequestration of the metal in the vacuole or in acidic intracellular vesicles. Plants

that lack both transporter proteins, therefore have decreased cadmium

resistance.

The role of AtClCa and AtClCd in heavy metal tolerance

75

INTRODUCTION

Heavy metals are elements that have an atomic weight between approximately 63-

200 Daltons. Over 50 elements have been classified as heavy metals, 17 of which are

very toxic and relatively accessible. They are naturally occurring minerals that are

found throughout our natural environment. Contamination of soil with heavy metal is

a serious worldwide problem both for human health and agriculture (Gairola et al.,

1992; Mazess and Barden, 1991 and Ryan et al., 1982). Metal toxicity interferes with

cellular activity by several mechanisms: displacement of essential cations, induction

of oxidative stress, and direct interaction with proteins. Cadmium is a toxic heavy

metal that enters the environment, and also the food chain, through industrial

processes and phosphate fertilizers (Pinto et al., 2004). In plants, cadmium is taken up

easily by the roots of many plants species, where it can be loaded into the xylem and

transported to the leaves. Cadmium has a 2-20 times higher toxicity than most of the

other heavy metals (Jagodin et al., 1995). Cadmium toxicity is associated with growth

inhibition and imbalances in many macro and micronutrient levels. Cadmium toxicity

symptoms are more apparent in the root than the shoot, as the accumulation of Cd2+ in

the root is significantly higher than in the shoot (Breckle, 1991). In plants a low

concentration (5-10 μM) of Cd2+ reduces chlorophyll content and the photosynthetic

yield in Brassica napus (Baryla et al., 2001; Larsson et al., 1998), displaces Ca2+ in

the photosystem II (Faller et al., 2005), is negatively affecting in respiration (Greger

and Ogren, 1991; Reese and Roberts, 1985) and inhibits water transport (Barcelo and

Poschenrieder, 1990). Cadmium inhibits almost all enzymes of the Calvin cycle in

pigeon pea and wheat plants (Sheoran et al., 1990; Malik et al., 1992). Cadmium also

induces the generation of reactive oxygen species (ROS), resulting in the unspecific

oxidation of proteins and membrane lipids and DNA damage (Dean et al., 1993;

Ames et al., 1993), inhibits germination (Sarath et al., 2007) and suppresses root cell

elongation (Stohs and Bagchi, 1995; Schutzendubel et al., 2001). Uptake studies

suggest that transport of Cd2+ into the cytoplasm and vacuole might depend on both

active and passive transport systems (Costa and Morel, 1993; Costa and Morel, 1994;

Hall, 2002; Hart et al., 1998; Salt and Wagner, 1993).

Changes in the cytoplasmic calcium concentration are used by the cell as an

almost universal second messenger system for many signals. Disturbance of calcium

Chapter 5

homeostasis and displacement of calcium have been suggested as possible

mechanisms of Cd2+, Zn2+, Cu2+, or Al3+ toxicity (Kinraide et al., 2004). For instance,

the vacuolar Ca+2/H+ antiporter CAX2 in Arabidopsis is able to transport Ca2+, Cd2+

and Mn2+ (Hirschi et al., 2000). Cadmium competes with Ca2+ at both the Ca2+-

channel (Nelson, 1986) and at intracellular Ca2+ binding proteins (Rivetta et al.,

1997). Exposure to cadmium resulted in a decrease of the calcium content in different

plant species (Gussarson et al., 1996; Sandalio et al., 2001). This competition between

calcium and cadmium seems to work both ways: increasing the external calcium

concentration alleviates the effects of cadmium, an effect that is assumed to result

from the competition for transporters between the two ions (Suzuki, 2005).

Anion channels are well documented in various tissues, cell types and membranes

of animals, protists and plants and current evidence supports a central role in cell

signaling, osmo-regulation, nutrient uptake and metal tolerance (Barbier-Brygoo et

al., 2000). Seven ClC genes have been identified in the Arabidopsis genome.

Subcellular localization is still largely putative, but AtClCa-b-c and g are assumed to

function in the tonoplast , AtClCd and AtClCf are localized to the Golgi membrane

and AtClCe is assumed to be targeted to the tylakoide membrane (De Angeli et al.

2007; Marmagne et al., 2007; Lv et al., 2009). Recently, the localization in the

tonoplast of AtClCa and its role as a NO3-/H+ antiporter was demonstrated (De Angeli

et al., 2006).

The aim of this study was to determine the link between Cd2+ toxicity on the one

hand and Ca2+ and ClC transporter-related heavy metal resistance on the other hand.

We show that Cd2+ at concentrations of 60 to 90 μM causes serious damage in the

primary roots of all genotypes. Ca2+ was able to alleviated Cd2+ reduction of root

growth in most genotypes, except in plants that were defective in both the AtClCa and

AtClCd transporter.


77

MATERIALS AND METHODS Plant material and culture conditions

Plant material and standard culturing conditions used are the similar to those

described in Chapter 2. To determine Cd2+ toxicity, the role of Ca2+ and pH in this,

plants were transferred to new plates with the same solid medium but supplemented

with the appropriate concentrations of CdCl2, ZnCl2, PbCl2, BaCl2, MgCl2 or CaCl2.

Also the pH of the Tris/Mes-buffered medium was adjused when needed.


Selection for single, double and triple mutants has been described in Chapter 2.

Cell imaging in primary root

Imaging was performed using a Nikon Coolpix 990 digital camera mounted on an

inverted optical microscope (CX41, Olympus, Tokyo, Japan) equipped with

objectives of 20× and 40× magnification. After 15 days of growth (after sowing) on

the vertically placed agar plate the primary root length and the distance between root

tip and first epidermal cell with visible root hair bulge (DFEH) were measured using

the microscope. For every experiment the average of the root length of at least 8

plants was calculated and every experiment was repeated twice.

Chapter 5

RESULTS

Reverse transcription PCR analyses

RT-PCR for Atclc single, double and triple mutant plants was carried out with

gene specific primers as described in chapter 2 and the bands were compared with the

tubulin product. RT-PCR confirmed the expression of AtClCa , AtClCb and AtClCd in

root tissue. In all mutant genotypes the absence of transcripts confirmed that the T-

DNA insertions all resulted in null alleles (Figure 1).

Figure 1. The absence of expression of the AtClCa, AtClCb and AtClCd genes in the double and triple mutant lines. The five genotypes were analysed using the primers shown in table 1 of chapter 2. The Tubulin transcript levels of the five genotypes are shown as a loading control.

Cd2+, Pb2+ and Zn2+ inhibits primary root growth in Atclc mutant plants

Exposure of 7 days old seedling to 90 μM Cd2+ resulted after 8 days of further

growth in strongly reduced primary roots (Figure 2a). Exposure to 90 μM ZnCl2

(Figure 3) or lower CdCl2 (Figure 2a) concentrations only weakly inhibited primary

and lateral root growth. In all treatments, root growth was not significantly different

between single, double and triple mutant and wildtype plants.


79

Figure 2. The inhibition of growth of the primary roots of the wildtype Arabidopsis and the 7 mutant genotypes by Cd2+ and the alleviation of the inhibition by Ca2+. a: Concentration dependence of Cd2+ inhibition in absence and the presence of 30 mM CaCl2. b: Ca2+ concentration dependence of the alleviation of the inhibition of root growth by 90 μM Cd2+. Datapoints are the average of 3 experiments and the error bars indicate the standard deviation.

Alleviation of cadmium toxicity by Ca2+ is different between wildtype and

Atclcad double mutants

Based on the results of Suzuki (2005), that increased external Ca2+ reduces the

effects of Cd2+ we tested the effect of 30 mM CaCl2 in plants exposed to toxic levels

of cadmium. The application of CaCl2 to Cd2+-stressed plants, restored the growth of

the primary root in all genotypes, except in two. In the Atclcad double mutant and in

the triple mutant Atclcabd the primary and lateral root was significantly shorter than

in the other genotypes (Figure 2a). The effect of calcium is dose-dependent and with

90 μM Cd2+ the Km for calcium is about 1 mM (Figure 2b). The restoration of root

growth in plants exposed to cadmium seems to be a calcium-specific effect. Addition

of BaCl2, cholinchloride or MgCl2 did not show any positive effect on the root growth

of plants that were treated with 90 μM CdCl2 (Figure 4).

.

Figure 3. Inhibition of root growth by Zinc and the absence of allevetation by Ca2+ in the wildtype Arabidopsis and the 4 multiple mutant genotypes. Datapoints are the average of 3 experiments and the error bars indicate the standard Deviation

Chapter 5

In animal cells a relation between Zn2+-toxicity and anion channel function had

been demonstrated (Duffield et al., 2005 ). To test whether a similar relation exists in

plants, we studied the effects of Ca2+ on Zn2+-treated wildtype and Atclc mutant

plants. Figure 3 shows that, in contrast to Cd2+-treated plants, calcium does not

change the effect of zinc on root growth, nor does the genotype of the plant influence

the extent of the inhibition.

Figure 4. Influence of different cations on the inhibition of root growth in the wildtype Arabidopsis and the 4 multiple mutant genotypes by Cd2+. The used concentrations of CaCl2, BaCl2 CholineCl and MgCl2 are 30 mM. Datapoints are the average of 3 experiments and the error bars indicate the standard deviation. Effects of Cd2+ on root morphology

In plants exposed to cadmium the morphology and color of the root tip changes

and the shape of the cells in the elongation zone is distorted (Suzuki, 2005). In our

experiments, exposure to 90 μM Cd2+ also leads to deformation of the root tip and a

change in morphology of the cells along the root rip. In cadmium-treated roots the

diameter of the root tip is decreased and the color of the cells much darker (Figure 5).

At lower concentrations of Cd2+ (30-60 μM) the diameter of the primary root also

decreased and initiation of lateral root growth was still higher, but the shape and color

of the cells in the root tip were not different from the controls. The experiment shown

in figure 5 confirms that a high concentration of Cd2+ causes serious damage to the

cells at the border between the elongation and meristematic zone.


81

Figure 5. Phenotypes of the roots of the Arabidopsis wildtype and the four multiple mutants when grown on media with Cd2+ or Cd2+ and Ca2+. The bar in the upper left photo indicates 0.5 millimeter.

These effects of cadmium poisoning can also be alleviated by increasing the

external calcium concentration (Figure 5, right panels). In all genotypes the

morphology of calcium-treated roots is not significantly different from the controls.

For comparison we also exposed the plants to a high concentration of lead. 700 μM

Pb2+ does induce changes in the diameter and color of root, but does not affect the

shape of the cells in the tip of the primary root cells in same way as 90 μM Cd2+ does

(data not shown).

Relation between Cd2+, Ca2+ and pH condition in primary root growth

In Chapter 4 we showed that root growth in the Atclca and Atclcb mutants is more

sensitive to pH than in the wildtype plants. Since it is the combination of these same

mutated genes in which Ca2+ has a reduced capability to alleviate cadmium-toxicity

we tested the effect of pH on cadmium toxicity in the different genotypes (Figures 6

and 7).

Chapter 5

Figure 6. The pH dependence of Cd2+ inhibition of root growth and the pH dependence of Ca2+ alleviation of this inhibition in wildtype Arabidopsis and the four multiple mutants. a: pH of the growth medium was 5.8 b: pH of the growth medium was 6.2. The length of the reference in the pictures in 2.5 cm. At pH 5.8 we obtained a result comparable with the data presented in figure 2. At the

higher pH the mutations in both AtClCa and AtClCd genes resulted in a stronger pH-

induced reduction of root growth. Curiously, at pH 6.2 the addition of 30 mM CaCl2

resulted in a complete reversal of the effect of Cd2+. At pH 6.2 no statistically

significant difference was found between control, the addition of only Ca2+ and the


83

addition of both Ca2+ and Cd2+. The difference between the genotypes was present

also in the control situation, indicating that at pH 6.2 the effect of pH on root growth

of the genotypes is dominating the response.

Figure 7. The pH dependence of Cd2+ inhibition of root growth and the pH dependence of Ca2+ alleviation of this inhibition in wildtype Arabidopsis and the four multiple mutants. Plants were grown in the presence of either 90 μM CdCl2, 30 mM CaCl2 or the combination of both salts. a: pH of the growth medium was 5.8 b: pH of the growth medium was 6.2

Chapter 5

DISCUSSION

In order to understand the relationship between heavy metals and anion

transporters in plant cells, we made single, double and triple anion channel mutants in

Arabidopsis thaliana (Figure 1). With these genotypes we show, firstly, specific

effects of cadmium on primary root growth and development, that cannot be

mimicked by the other heavy metals tested (zinc and lead). Secondly, the effects of

cadmium can be reversed by increasing the external calcium concentration. Thirdly,

we show that this effect of calcium depends on the presence of AtClCa or AtClCd.

The effect of cadmium on roots is different from the effect of other heavy metals

In plants exposed to cadmium the accumulation in the roots will be higher than in

the shoot and therefore phytotoxic effects will be more apparent in the root (Breckle,

1991). Although all three heavy metals tested resulted in inhibition of root growth, the

mode of action of cadmium differs from that of zinc and lead. The effects of lead do

resemble those of cadmium, as both result in a narrow root tip and a darkening of the

cells. However, cell deformation of the cells in the root tip that are characteristic for

exposure to sub-lethal concentrations of cadmium, were not observed after exposure

to lead. In Arabidopsis root that cell death first appeared in around meristematic and

then in elongation zone of root, where influx of Cd2+ caused cell death and inhibited

primary root growth (Suzuki, 2005).

While no differences between the genotypes, concerning heavy metal sensitivity,

were observed, a difference was observed when the capability of Ca2+ to alleviate the

Cd2+ reduction of root growth was studied. Ca2+ was unable to alleviate the Cd2+

effects in the mutant plants lacking functional AtClCa and AtClCd. The reduction of

root growth induced by Zn2+ is equal in all genotypes and is insensitive to calcium.

We interpret this effect of calcium as an increased sensitivity of the genotypes Atclcad

and Atclcabd for Cd2+.

Plant cells have developed a variety of mechanisms to protect cells from heavy

metals. One of them is the accumulation of soluble phenolic compounds in the cells,

resulting in protection of tissues against oxidative stress. (Yamamoto et al., 1998;

Schutzendubel et al., 2001; Suzuki, 2005), which is visible as the dark discoloration in

Cd2+ or Cu2+ exposed root tips. Another is exclusion and/or sequestration.


85

In effects of calcium on cadmium toxicity: differential effect of Ca2+on Atclcad

double mutants

Cadmium mainly enters the root in the first few 1-1.5 mm behind the root tip and

the influx is significantly less, further up the root (Pineros et al., 1998; Arduini et al.,

1996). This is also the normal pattern of uptake for plant nutrients and several studies

indicate that it is likely that Cd2+ enters the plant, in a competitive way, through the

same transporters that are involved in nutrient uptake. Exposure to cadmium can

result in a decrease in the content of Ca, Zn, Cu, Mn and Fe in pea leaves (Rodriguez-

Serrano et al., 2009). Cadmium uptake could be competitively inhibited by other

cations and by Ca2+-channel blockers (Blazka and Shaikh, 1992; Clemens, 2006).

Active and passive transport systems have been reported for Cd2+ in roots of several

plant species (Cataldo et al., 1981; Godbold, 1991; Gosta and Morel, 1993 and 1994;

Hart et al., 1998). Non-essential heavy metals might be transported via nutrient

transporters or channels that are not completely selective (Clemens et al., 1998). It has

been observed that cadmium not only competes with calcium for calcium transporters,

but also for intracellular Ca2+-binding proteins (Rivetta et al., 1997) and at plasma

membrane (Kinraide, 1998). Alleviation by calcium of Cd2+ toxicity by reducing the

Cd2+ uptake and accumulation, have been reported in radish (Rivetta et al. 1997),

tobacco (Choi et al., 2001), rice roots (Kim et al., 2002) and Arabidopsis seedlings

(Suzuki, 2005). Also, in Arabidopsis thaliana AtHMA1 functions as a Ca2+/ heavy

metal pump (Moreno et al. 2008). Analysis of the interaction between calcium and

cadmium in wildtype and all mutant plants the extent of the effect of Ca2+ on Cd2+-

toxicity is related the concentrations of Cd2+ and Ca2+. Our results confirm this

conclusion: 30 mM Ca2+ almost completely compensated the toxic effect of 30 and 60

μM Cd2+ (except for AtClCad and AtClCabd in 60 μM Cd2+). But at 90 μM Cd2+ the

alleviation by 30 mM Ca2+ was not complete. Furthermore, in 90 μM Cd2+, 0.5 – 1

mM of Ca2+ reduces toxicity by 1/3 to 2/3 and 2.5-30 mM Ca2+ reduces the effect of

cadmium by more than 2/3 (Figure 2b).

Our results showed that, of the different cations tested (BaCl2, MgCl2 , CaCl2 and

cholinchloride), only addition of CaCl2 alleviated cadmium-induced root growth

inhibition. This rules out that the antagonism between cadmium and calcium depends

on the occupation of extracellular binding sites, as for such a mechanism one would

expect also a positive effect of the other divalent cations.

Chapter 5

The most intriguing result in our study is the differential effect of calcium on the

cadmium toxicity in the Atclca and Atclcd mutants. Increasing the calcium

concentration in the double mutant AtClCad and triple mutant AtClCabd treated with

Cd2+, alleviated root growth significantly less than in other mutants and wildtype

plants (Figure 2). Several reports have made a connection between anion transporters

and heavy metal translocation. Heavy metals are transported across cell membranes

by a number of complex mechanisms. In animals metal transport into cells is sensitive

to the anion channel blocker DIDS (Simons, 1986; Lou et al., 1991). For animal cells

it has been proposed that heavy metals can cross the membranes via the anion channel

in the form of anionic complexes with carbonate, bicarbonate, hydroxyl, or chloride

ions (Foulkes, 2000).

Several previous investigations have demonstrated the important role of Ca2+ for

root elongation, even in the absence of metal toxicity (Demidchik et al., 2002;

Hanson, 1984; Kinraide, 1998). In plant guard cells (Schroeder and Hagiwara, 1989;

Hedrich et al., 1990; Allen et al.,1999; Blatt, 1999 and Leonhardt et al., 2004) and in

Arabiopsis thaliana suspension cells (Trouverie et al., 2008) anion channels are Ca2+-

sensitive and activated by transient increases of [Ca2+]. In Arabidopsis thaliana

hypocotyl protoplasts, activation of anion channels is directly dependent to the

calcium concentration at the cytosolic site of the plasma membrane (Lewis et al.,

1997).

Furthermore, reactive oxygen species (ROS), such as H2O2, are involved in

signaling pathways through the activation of plasma membrane calcium channels (Pei

et al., 2000; Murata et al., 2001, Kwak et al., 2003; Trouverie et al., 2008). One could

hypothesize that activation of the anion current would result from the activation of

calcium channels. Therefore, a simple explanation for the activation of anion channels

by oxidative stress and calcium application would rely on the ability of heavy metal-

induced increased ROS concentrations to promote Ca2+ influx. However, the even

simpler hypothesis that cadmium toxicity is prevented by external calcium through

competition for transporters and thus reduction of the intracellular [Cd2+], is a good

possibility.

However, in both explanations the mechanistic role of AtClCa and AtClCd is not

obvious. Here we present a model for the role of these anion transporter proteins in

cadmium-tolerance (Figure 8). AtClCa and AtClCd are located on endomembranes,

have H+/anion antiporter activity and have the highest expression levels in the


87

expansion and transition zone of the root (Lv et al., 2009). In yeast, mutants of gef1,

an anion transporter located in the trans-Golgi vesicles, fail to properly regulate pH

and are more sensitive to heavy metals. Heterologous expression of AtClCa, AtClCc

or AtClCd could at least partly complement gef1, and restore heavy metal resistance

(Gaxiola et al. 1998).

Figure 8. Model for the role of ClC transporter proteins in the sequesteration of cadmium in intracellular compartments (e.g. the vacuole). A: Protons are actively pmped into the vacuole, resulting in a pH gradient and a tonoplast potential (positive inside), the activity of the ClC anion/proton antiporters proteins reduces the tonoplast potential and increases the concentration of anions in the tonoplast. This facilitates the the accumulation of positively charged Cd2+ in the vacuole. B: When the ClC anion/proton antiporters are either inactive or absent this leads to an excess of positive charges and an acidic pH in the vacuole. This situation reduces sequesteration of the Cd2+ in the vacuole.

We propose that in Arabidopsis thaliana AtClCa and AtClCd have a similar

function as a H+- and electrical shunt for the V-type ATPase and the heavy metal

transporter, respectively. Functional AtClCa and AtClCd proteins facilitate a high

accumulation of heavy metal in either the vacuole and/or acidic vesicles. The

observation that only the double mutant is more sensitive implies that AtClCa and

AtClCd have overlapping functionality. One possibility is that both are present in the

membrane of the same compartment and thus are truly redundant. Another option is

that they are localized in different compartments, but that heavy metal sequestration

occurs in both compartments, viz. the acidic trans-Golgi vesicles and the vacuole. In

this model the differential effect of Ca2+ on cadmium toxicity is based on two distinct

mechanisms of heavy metal resistance: exclusion, which is aided by addition of

calcium, and sequestration, which is facilitated by active AtClCa or AtClCd. Neither

gives full protection when exposed to 90 μM Cd2+, but in combination they can

restore full root growth and development.

Chapter 5

ACKNOWLEDGEMENTS

This work was supported in part by a grant from the Ministry of Science, Research

and Technology of the Islamic Republic of Iran.

89

Chapter 6 General discussion and conclusions Hossein Moradi1,2, Theo Elzenga1 and Frank Lanfermeijer1

1 Department of Plant Biology, University of Groningen, 9750 AA Haren, The Netherlands 2 Department of Agronomy and Plant Breeding, Sari Agricultural Sciences and Natural Resources

University, Iran

Chapter 6

Functional redundancy In the introduction we stated that we want to explore the position of the ClC

proteins in the complex network of membrane transport and solute fluxes. This study

shows that this is a complicated task. First of all, the most important problem seems to

be the absence of obvious phenotypes for single mutants. It requires creativity of the

scientist to generate conditions that evoke phenotypes. However, even the breeding of

double and triple mutants is no easy road to success (Chapter 2). Appearently, the ClC

channels exhibit high functional redundancy.

We started from the rational that the ClC proteins have a clear function in either

Cl- or NO3- homeostasis (Chapter 2 and 3). However, the absence of clear phenotypes

when, for instance, seeds were germinated in the presence of high concentrations of

these anions might indicate that their role in the homeostasis of those ions is limited.

Plants apparently have developed other transporter systems for these functions.

Our results more clearly indicate that ClC proteins play an important role in

energizing the membrane and pH homeostasis. Energizing the membrane and pH

homeostasis depend protons fluxes and currents carried by other ions. For generating

a large [H+] gradient, necessary for a high capacity of secondary active transport of

solutes, the electrical potential difference across the membrane has to be kept low.

The electrical component of the proton pumping ATPase activity therefore has to be

short-circuited. This shorting of the proton pump could be accomplished by any kind

of current. It does not matter if it’s carried by Cl- or NO3- or even K+ (but then in the

opposite direction). Although the ClC proteins can have distinct functions, for

instance being either a Cl- or an NO3- transporter, their electro-physiological

characteristics can still make them redundant as both can act as an electrical shunt in

the generation of the proton-motive force (Chapter 4 and 5). The situation is probably

even more complex as a shunt function can also be performed by a system not related

to the ClC proteins, for instance, by a K+ channel. Hence, this suggests that in order to

study the role of ClC proteins in energizing the membrane, the experiments should be

highly defined and controlled. Composition of experimental media should be simple

and the potential of alternative currents to occur should be limited. All electro-

physiological tools should be used and, if possible, ClC-mediated fluxes should be

studied in plants were alternative currents can be excluded.

The second role of ClC proteins for which we found indications, is in the

accumulation in osmotically active solutes. Osmotically active solutes can be any

General discussion and conclusion

91

soluble ion or compound. A cell can accumulate other solutes if one of the normally

used solutes (Cl- or NO3-) is not available or can not be used (for instance in the

absence of AtClC proteins). Hence, in this case complementation of the function can

also be expected outside the group of ClC proteins: for example by malate or sugar

transporters.

A nice example of this type of redundancy is shown in Chapter 5. In this chapter it

is shown that Ca2+ can not alleviate Cd2+-induced reduction of root growth if AtClCa

and AtClCd are both absent. The currently favored model for explaining our

observation is, that Cd2+ toxicity is countered in two ways: 1) Ca2+ protects the plant

by competing with Cd2+ for essential sites in enzymes and transporters and 2) Cd2+ is

sequestered in internal compartments (vacuole), rendering them harmless. We propose

that in the latter mechanism ClC proteins play a role as shunts by allowing generation

of a PMF across intracellular membranes and the transport of Cd2+ across these

membranes (Chapter 5). In Chapter 1 (Table 1) it is suggested that AtClCa is a

H+/NO3- and AtClCd is a H+/Cl- antiporter. The fact that you need to remove both

transporters demonstrates that both proteins can be used as a shunt and complement

each other. If one of the two ClC proteins is missing, either the Cl- or the NO3- ions

can still be used to compensate the movement of positive charges (H+) by the primary

proton pumps. Thus, only in the case where both ClC proteins are removed, there are

apparently no transporters (or their charged substrates) present that can take over the

function as shunt. It would be interesting to study the effect of Ca2+ on Cd2+ toxicity

when the various genotypes are grown either on low Cl- or low NO3- media.

Root growth as studied in Chapters 2, 3 and 4 showed also that the main functions

of the ClC proteins are electrical circuits, which allow the generation of a PMF to

drive the uptake of osmotically active solutes or stimulate the capacity to acidify the

apoplast and thus facilitate cell expansion.

Intracellular localization of the ClC proteins

Although we do not need the exact localization of the ClC proteins in the model

proposed in Chapter 5. It remains an important issue for understanding the roles of

these proteins. And, as proposed above, if ClC proteins can functionally be replaced

by proteins that are not related to them, localization can become an important issue.

As suggested by the articles of Moore and Murphy (2009) and Millar et al. (2009)

localization studies are difficult and need a careful approach. Presently only the paper

Chapter 6

of De Angeli et al. (2006) seem to meet some of the proper standards. In that study

localization experiments on AtClCa are convincingly combined with physiological

measurements aimed at elucidating the function (patch clamp measurements).

Final conclusions The study of ClC proteins is a complicated one. They seem to play a role in

different processes (osmo-regulation, detoxification, cell expansion, and maybe

more). Roles that are so important in plant cells, that several protein systems exists

which can fulfill these roles next to the ClC proteins. Functional studies on these

proteins require, next to creativity of the researcher, very specific conditions in the

experiments (for instance Ca2+ alleviation of Cd2+ effects) to observe phenotypes.

Several important issues remain to be studied: the role of the two CBS domains and

the role of ATP binding in the functioning of ClC proteins as shunts and osmo-

regulators. Also important for plants is the strictness of the duality of ClC proteins as

specific Cl- or specific NO3- transporters and the strictness of the duality as channels

or H+-cotransporters. The study of the ClC protein family will remain a challenge for

researchers in the coming years and, very likely, will yield unforeseen and surprising

results.

93

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SUMMARY

In higher plants anion channels, present in various tissues and cell types, play

importants roles in signaling pathways leading to the adaptation of plant cells to

abiotic and biotic stresses, in the control of metabolism, and in the maintenance of the

electrochemical gradients across membranes. One of the an protein families with

anion channel function, is the Chloride Channel (ClC) family. First characterized in

Torpedo marmorata, these proteins appear now to be ubiquitously present. Seven ClC

genes are identified in the Arabidopsis genome. The main aim of this research was to

physiologically characterize Arabidopis thaliana ClC proteins and to acquire

knowledge on the position of ClC proteins in the complex network of membrane

transport and solute fluxes. We used a reverse genetics approach with T-DNA knock-

out mutants of three of the 7 ClC-genes that are present in the genome of Arabidopsis

thaliana: AtClCa, AtClCb and AtClCd.

In chapter 1 we review the current state of the art on ClC transport proteins and

relate the knowledge obtained from bacterial and non-plant systems with the

Arabidopsis ClC proteins. Predictions and remarks, concerning function and

localization of plant ClC transporters, are made.

In chapter 2 the protocol of obtaining the three double mutants and the single

triple mutant that can be made with the three single knock-out mutants, is described.

The seven mutants are preliminary characterized by testing the effect of the mutation

on seed germination, vegetative growth and ion fluxes in root and leaf tissue as

measured by the Micro-Electrode Ion Flux Estimation (MIFE).

In chapter 3 the MIFE technique was expanded with a flow-through system

which allowed a controlled and gentle exchange of the measuring solution during an

experiment. Ion fluxes from Atclcd mutant plants, which lack the AtClCd protein,

were compared with those from wildtype plants. The results of these experiments

indicate that AtClCd functions as an anion/H+ antiporter and might be involved in the

accumulation of both Cl- and NO3- in the vacuole.

In chapter 4 a root growth phenotype is described for mutant plants in which

either AtClCa or AtClCd are knock-out by a T-DNA insertion. At pH 6.2 root growth

in those mutant plants is impaired, compared with wildtype and the Atclcb mutant

plants. We measured the proton fluxes along the root from root tip till the first root

hairs at different pH values. Additionally root growth was characterized and

quantified. A model for the interaction between endomembrane anion/H+ antiporters,

plasma membrane proton fluxes and cell expansion is discussed.

In chapter 5 we study the effect of heavy metal treatments on the 7 mutant plants

(single, double and triple mutants). No differences were observed between the root of

the mutants and wildtype for cadmium, zinc and lead. Cadmium toxicity could be

alleviated by increasing the external calcium concentration. This effect of high

external calcium is not observed in the plants lacking both AtClCa and AtClCd. The

double mutant Atclcad and the triple mutant Atclcabd therefore seem to be more

sensitive to cadmium toxicity. Based on these results a role of AtClCa and AtClCd in

cadmium detoxification is suggested and a mechanism is proposed.

In chapter 6 all our result are placed in the context of the earlier published

functions and localization of ClC transporter proteins. It is suggested that the main

role of the ClC proteins is to function as electrical short circuits, which plays an

important role in energizing the membranes. A role in NO3- and Cl- homeostasis

seems of minor importance. Another important issue is the intracellular localization of

these proteins. Knowledge on the whereabouts of these proteins in the cell has

important implications on their functions. Although the results, published in the

present literature are in agreement with the ideas of the function of the ClC proteins,

care must still be taken with the interpretation of the fluorescence data on the

localization of the ClC proteins. The published data seems to lack important controls.

109

SAMENVATTING

Anion-kanalen komen voor in praktisch alle cel- en weefseltypen van hogere

planten. Zij hebben verschillende, belangrijke functies: in signalerings-routes die

leiden tot aanpassing aan abiotische of biotische stress factoren, in het metabolisme en

in het handhaven van elektrochemische gradiënten over membranen. Eén van de

eiwitfamilies met een anion-kanaalfunctie is de Chloride Channel (ClC) familie. Het

eerste lid van deze familie werd gevonden in het elektrische orgaan van de

Gemarmerde Sidderrog (Torpedo marmorata), maar blijkt nu alom vertegenwoordigd

in dieren, planten en bacterien. In het genoom van de Zandraket (Arabidopsis

thaliana) zijn zeven ClC genen geïdentificeerd. Het doel van dit onderzoek was om de

ClC eiwitten in Arabidopsis fysiologisch te karakteriseren en om kennis te vergaren

omtrent de positie van ClC eiwitten in het complexe netwerk van membraantransport

en ionstromen. We hebben een ‘reverse genetics’ benadering gebruikt met T-DNA

knock-out mutanten van drie van de zeven ClC genen die aanwezig zijn in het

genoom van Arabidopsis: AtClCa, AtClCb en AtClCd.

In hoofdstuk 1 bespreken we de huidige stand van zaken omtrent de ClC

transporteiwitten en wordt de kennis vergaard uit bacteriële en niet-plant systemen

gerelateerd aan de ClC eiwitten in Arabidopsis. De mogelijke functies en de

lokalisatie van de ClC transporteiwitten in planten worden behandeld.

In hoofdstuk 2 wordt het protocol waarmee de drie dubbel-mutanten en de ene

drie-dubbel mutant verkregen kunnen worden uit de drie enkele knock-out mutanten

(AtClCa, AtClCb en AtClCd) beschreven. In een globale karakterisering werden de

effecten van de mutaties in deze zeven mutanten getest op de ontkieming en

vegetatieve groei. Verder werden de ionstromen in wortel- en bladweefsel bepaald

middels de Micro-Electrode Ion Flux Estimation (MIFE) techniek.

In hoofdstuk 3 werd de MIFE techniek uitgebreid met een zogeheten doorstroom-

systeem waarmee de meetoplossing op een gecontroleerde en subtiele manier

uitgewisseld kon worden tijdens het experiment. Ionenstromen van Atclcd mutanten,

waarin het AtClCd eiwit ontbreekt, werden vergeleken met wildtype planten. De

resultaten van deze experimenten geven aan dat AtClCd functioneert als een anion/H+

antiporter en wellicht betrokken is bij de ophoping van zowel Cl- als NO3- in de

vacuole van plantencellen.

In hoofdstuk 4 wordt het fenotype van de wortel beschreven voor gemuteerde

planten waarin zowel AtClCa of AtClCd middels een T-DNA insertie zijn

uitgeschakeld. Bij een pH van 6.2 wordt wortelgroei bij deze mutanten geremd

vergeleken met het wildtype en de AtClCb mutant. We hebben de protonenstroom

langs de wortel, van het wortelmutsje tot de eerste wortelhaar, gemeten bij

verschillende pH waarden. Uit kwantificatie van verschillende aspecten van de

wortelgroei kan worden afgeleid dat de mutaties de strekkingsgroei van de cellen

beinvloeden. Een model voor de interactie tussen endomembraan anion/H+

antiporters, protonenstroom over het plasmamembraan en celexpansie worden

bediscussiëerd.

In hoofdstuk 5 behandelt het effect van blootstelling aan zware metalen in de

zeven mutanten (de enkele, dubbele en driedubbele mutanten). Er werden geen

verschillen waargenomen tussen de wortelgroei van de mutanten en het wildtype

behandeld met cadmium, zink en lood. Cadmium toxiciteit kon worden verminderd

door de externe calcium concentratie te verhogen. Dit effect van een verhoogde

externe calcium concentratie werd niet waargenomen in planten waarin zowel AtClCa

als AtClCd ontbreken. De dubbele mutant Atclcad and the triple mutant Atclcabd

lijken daardoor meer gevoelig te zijn voor cadmium. Gebaseerd op deze resultaten

wordt een rol van AtClCa en AtClCd in de detoxificatie van cadmium gesuggereerd

en een mechanisme wordt voorgesteld.

In hoofdstuk 6 worden al onze bevindingen geplaatst in de context van eerder

gepubliceerde functies en lokalisaties van de ClC transporteiwitten. Een belangrijke

rol van de ClC eiwitten is het kortsluiten van de elektrische component van de

protonenpomp in de tonoplast, waardoor deze de mogelijkheid krijgt om een sterk

gradiënt op te bouwen in de protonen concentratie aan weerzijde van de tonoplast.

Een rol in de NO3- en Cl- homeostase van de cel lijkt van minder groot belang. Kennis

over de de intracellulaire lokalisatie van deze eiwitten in de cel heeft belangrijke

implicaties voor hun functies. De ideeën die in dit proefschrift over de functies van de

ClC eiwitten worden beschreven, zijn grotendeels in overeenstemming met de

111

resultaten die al eerder gepubliceerd zijn. Echter, voor wat betreft de lokalisatie van

deze eiwitten lijkt dat niet het geval. De studies waarin transporteiwitten worden

geassocieerd aan een specifieke membraan in de cel zijn tot nu toe niet adequaat

uitgevoerd. Definitieve conclusies zullen pas kunnen worden getrokken wanneer bij

lokalisatie studies de controle of artefacten serieus plaatsvindt en wanneer ze worden

gecombineerd met fysiologische metingen aan de betrokken membranen.

چکيده

بخشهای مختلف گياهان وغشا های پروتئينهای آنيون ترانسپورتر در ،در گياهان عالی

نقش در چرخه سلولی از طريق کنترل اين پروتئينها با ايفا .سلولی ديده شده استمختلف اطراف سلول باعث سازگاری سلولهای گياهی 1ميايیمتابوليسم سلولی وحفظ شيب الکتروشي

يکی از مهمترين پروتئينهای اين 4کلرايد کانالها. می گردند 3و غيرزنده 2به استرسهای زندهاولين مطالعات در اين .خانواده به شمارمی روند که درانتقال آنيون به سلول نقش دارند

ام گرديد وبدنبال آن در جانوران وگياهان انج )5 ترپدمارمرات (پروتئينها بر روی نوعی ماهی.شناسايی ومطالعه شد

هدف اصلی دراين تحقيقات، بررسی ومطالعه مولکولی وفيزيولوژيکی آنيون کانالها در ونقش آنها درشبکه انتقال يونها ومواد دراطراف سلول تعريف 6گياه مدل ژنتيکی آرابيدبسيس

به 8وبا استفاده از انتقال دی ان ای باکتری 7ک معکوسدراين تحقيقات از روش ژنتي. گرديد 10جهش يافته سپس با استفاده ازگياهان. ايجاد گرديد9جهش درگياهان داخل ژنوم گياه ميزبان،

.که فاقد ژنهای موردنظربودن، نقش اين ژنها در چرخه سلولی بررسی شد

ساختمان کريستالی سلولی، با مروری بر انواع ترانسپورترها درغشائ-فصل اول ومولکولی توام با کارکرد پروتئينها ی آنيون ترانسپورتری در باکتريها وجانوران مورد

براساس اين مطالعات برخی از وظايف ، محل قرار گيری اين . مطالعه و بررسی قرار گرفت. گرديد 11ينیپروتئينها در گياهان وتقسيم بندی گياهان براساس ساختمان مولکولی آنها پيش ب

جهت بررسی وظايف برخی از ترانسپورترهای آنيونی در گياهان از طريق -فصل دوم

سپس آزمايشاتی . ميوتن ايجاد گرديد14و تری پل13، دبل12ژنتيک معکوس ابتدا گياهان سينگل، جوانه در رابطه با اثر فقدان اين ژنها درگياهان جهش يافته بر روی ذخيره آنيونها درگياهان

زنی بذر، مراحل مختلف رشدی گياه وجريان انتقال يونها دراطراف سلولهای ريشه وبرگ با مورد مطالعه قرار 15استفاده ازبرخی اندازه گيريهای ميکروالکترودی نظير دستگاه مايف

.گرفت

با انجام آزمايشات الکتروفيزيولوژيکی بر روی يکی ازژنهای مهم از آنيون-فصل سوم وبا تغيير و تحول در سيتم ثابت 16کانالها در آرابيدبسيس ،به نام پروتئين آنيون ترانسپورتر دی

اندازه گيری يونها دراطراف سلول گياهی به سيتم درحال گردش محلول دراطراف سلول، به طوريکه پروتئينهای آنيون .برخی ازکارکردهای اين پروتئين درسلول گياهی مشخص گرديد

جهت تبادل آنيون با پروتون معرفی 17ر گياه آرابيدبسيس به عنوان يک آنتی پرترکانال دی د.گرديد که در ذخيره يونهای کلرايد ونيترات در واکوئل سلولی مشارکت می کند

درغشاهای داخلی سلول )پروتون /آنيون( جهت مطالعه اثر متقابل آنتی پرترها -فصل چهارم

شائ سلولی با افزايش اندازه سلولی، ورود وخروج پروتونها به سلول وپمپهای پروتونی درغ .اندازه گيری شد18در قسمتهای مختلف ريشه گياهان جهش يافته وگياهان شاهد

نتايج حاصل ازاين آزمايشات منجربه ارائه الگوی جديدی دررابطه با اثرات پروتئينهای آنيون .ترانسپورتری برتغييرات حجم سلول گرديد

113

در جانوران، اثر 19 با توجه به نتايج اخيرارتباط آنيون کانالها با فلزات سنگين-فصل پنجم برخی فلزات سنگين نظير کادميم، روی وسرب بر روی گياهان فاقد برخی از ژنها ی آنيون

:ازميان نتايج حاصل ازآن، مهمترين نتايج شامل.کانالی مورد ارزيابی قرار گرفت تفاده از کل ادميم در اس رات سمی حاصل از ک اهش اث زان غلظت آن سبب ک ه مي سته ب سيم ب

.کند سلول گياه می گردد تاحدی که از کاهش رشد ريشه جلوگيری ويا جبران میای ای ا ، ژنه ون کاناله ف آني ای مختل ان ژنه ه ودی 20از مي ا در چرخ ه ديگرژنه سبت ب ن

سيم به ط .اثرات کادميم بيشترين نقش را ايفا می کنند وريکه نتايج حاصل از سميت زدايی کلوتن ای دی ل مي ه دب ش يافت ان جه ادميمی در گياه ميت ک ی 21برس وتن ای ب ل مي و تريپ

شان داد نسبت به ديگر گياهان جهش22دی ابرا . يافته وگياهان شاهد، اختالف معنی داری ن بناهی لول گي ادميم در س ی ک ه سميت زداي ن دو ژن در چرخ ايف اي ن برخی ازوظ مشخص ي

.گرديد

با جمع بندی نتايج حاصل از فصول قبلی، نتايج نهايی در رابطه با وظايف -فصل ششم به طوريکه پيشنهاد گرديد .ومحل قرارگيری پروتئينهای آنيون ترانسپورتری ارائه گرديد

قال که نقش اين پروتئينها در ايجاد انرژی پتانسيل مورد نياز در اطراف سلول نسبت به انتهمچنين اين پروتئين ها در غشا های داخلی . يونهای نيترات وکلرايد در سلول مهمتر است

. سلول درمقايسه با غشائ پالسمائئ نقش موثرتری ايفا می کنند

1- Electrochemical gradients 2- Biotic stress 3- Abiotic stress 4- Chloride Channel(ClC) 5- Torpedo marmorata 6- Arabidopsis thaliana 7- Reverse genetics 8- T-DNA 9- Mutation 10- Mutant 11- Predictions 12- Single mutant 13- Double mutant 14- Triple mutant 15- Micro-Electrode Ion Flux Estimation (MIFE) 16- AtClCd 17- Anti porter 18- Wildtype 19-Heavy metal 20- AtClCa 21- Atclcad 22- Atclcabd

ACKNOWLEDGEMENTS First and above all, I praise God, the almighty for providing me this opportunity and granting me the capability to proceed successfully. This thesis appears in its current form due to the assistance and guidance of several people. I would therefore like to offer my sincere thanks to all of them. Theo Elzenga, my esteemed promoter, my cordial thanks for accepting me as a Ph.D student, your warm encouragement, thoughtful guidance, critical comments, and correction of the thesis. I want to express my deep thanks to my esteemed copromotor Frank Lanfermeijer for the trust, the insightful discussion, offering valuable advice, for your support during the whole period of the study, and especially for your patience and guidance during the writing process. I would like to thank the members of the reading committee, Prof. dr. Jacques Hille, Prof. dr.Bert van Duijn and Prof. dr. Sergey Shabala for their excellent advises and detailed review during the preparation of this thesis. I thank Ger, for his advices and his friendly assistance with various problems all the time, especially for his help with the paperwork, translation of many Dutch letters and his help outside the lab. Mohamed and Fatma, I greatly appreciate your excellent assistance and your spiritual supports for me and my family during my PhD study. I will never forget the time that we were together and discussed about religion and politics. Mohamed, I am pleased that you accepted to be my paranymf. Wouter, Thank you for translating the summary into Dutch and you accepting to be my paranymph. Marten, thanks for your excellent technical assistance in the Lab, particularly for MIFE technique, and your kindly answers to my general questions. I am grateful to the secretary Jannie, for assisting me in many different ways and handling the paperwork. Thanks also to all the members of plant physiology, Luit, Ineke , Marten, Cordula, Fatma, Jan Henk, Hamid , Wouter, Jacquline, Ika, Muhammad, Aleksandra, Desiree, Ana, Bep, and Freek, for providing a good atmosphere in our department and for useful discussions. My roommates, Jacquline and Ika, thank you very much for making the atmosphere of our room as friendly as possible. Jacques, Paul, Marcel, Bert, Reza, Ijaz, Eelco, Kamran, Sujeeth, and Jos, thank you for allowing me to use your facilities at the department of Molecular Biology and for useful suggestions. I also appreciate the financial support of the Iranian Ministry of Science, Research and Technology, during my Ph.D study, particularly Abdollahi and Nazemi as the scientific representatives of the Iranian government in the Schengen countries. Thanks to all my close friends and Iranian community of Ph.D student and their esteemed families, Abdollah bigi, Eslami, Najafi, Ramzani, Ryazi, Sohani and Shyrzadian for the joyful gatherings and all their supports. Els Prins, Although we have lived far from our relatives, but communication with you, provided emotional atmosphere for us. Hereby, I would like to thank you for everything. I cannot finish without thanking my family. .

115

I warmly thank and appreciate my parents and my mother and father-in-law for their material and spiritual support in all aspects of my life. I also would like to thank my brothers, sister, and brothers and sisters-in-law, for they have provided assistance in numerous ways. I want to express my gratitude and deepest appreciation to my lovely sweet daughter, Narges, for her great patience and understandings and for being a nice Muslim girl. And finally, I know that you did not want to be named, a person that moved with me to the Netherlands and she lost her job because of it, My lovely wife, Dear Farveh, without your supports and encouragements, I could not have finished this work, it was you who kept the fundamental of our family, and I understand it was difficult for you, therefore, I can just say thanks for everything and may Allah give you all the best in return. Hossein Moradi September 2009, Groningen, the Nederlands

117

:اساتيد راهنما پروفسور دکتر تئو الزنگا

دکتر فرانک النفرماير

:کميته داوران پروفسور دکتر ژاکوس هيله

پروفسور دکتر سرجی شاباال

پروفسور دکتر برت وان دوئن

پژوهشهای مندرج در اين رساله با حمايت مالی وزارت علوم،تحقيقات و فناوری جمهوری اسالمی ايران در دپارتمان فيزيولوژی گياهی ودپارتمان

.زيست مولکولی گياهی دانشگاه گرونينگن هلند،انجام شده است

دانشگاه گرونينگن هلند

دانشکده رياضيات وعلوم زيستی

ه دکتری در علوم زيستیرسال

تحت عنوان

مطالعه پروتئينهای آنيون ترانسپورترآنيون ترانسپورتری در آرابيدبسيسبررسی کارکردهای ميوتنهای

در جلسه دفاعيه مورخ1388 شهريور 30دوشنبه

11ساعت

حسين مرادی

· vi the research reported in this thesis was financially supported by a grant from the ministry...

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