Functional Characterization of - Plant and Cell Physiology

Functional Characterization of Arabidopsis Calreticulin1a: A Key Alleviator

of Endoplasmic Reticulum Stress

Anna Christensen1, Karin Svensson

2, Staffan Persson

3,*, Joanna Jung

4, Marek Michalak

4,

Susanne Widell2and Marianne Sommarin

1, 5

1 Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, PO Box 124, SE-22100 Lund, Sweden2 Department of Cell and Organism Biology, Biology Building, Lund University, Solvegatan 35, SE-22362 Lund, Sweden3 Max-Planck-Institute of Molecular Plant Physiology, Am Muehlenberg 1, DE-14476 Potsdam, Germany4 Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G2H7, Canada5 Department of Plant Physiology, Umea Plant Science Centre, Umea University, SE-90187, Umea, Sweden

The chaperone calreticulin plays important roles in

a variety of processes in the endoplasmic reticulum (ER) of

animal cells, such as Ca2þ signaling and protein folding.

Although the functions of calreticulin are well characterized

in animals, only indirect evidence is available for plants.

To increase our understanding of plant calreticulins we

introduced one of the Arabidopsis isoforms, AtCRT1a, into

calreticulin-deficient (crt�/�) mouse embryonic fibroblasts.

As a result of calreticulin deficiency, the mouse crt�/� fibro-

blasts have decreased levels of Ca2þ in the ER and impaired

protein folding abilities. Expression of the AtCRT1a in mouse

crt�/�fibroblasts rescued these phenotypes, i.e. AtCRT1a

restored the Ca2þ-holding capacity and chaperone functions

in the ER of the mouse crt�/�fibroblasts, demonstrating

that the animal sorting machinery was also functional for

a plant protein, and that basic calreticulin functions are

conserved across the Kingdoms. Expression analyses using

a b-glucuronidase (GUS)–AtCRT1a promoter construct

revealed high expression of CRT1a in root tips, floral tissues

and in association with vascular bundles. To assess the impact

of AtCRT1a in planta, we generated Atcrt1a mutant plants.

The Atcrt1a mutants exhibited increased sensitivity to the

drug tunicamycin, an inducer of the unfolded protein response.

We therefore conclude that AtCRT1a is an alleviator of the

tunicamycin-induced unfolded protein response, and propose

that the use of the mouse crt�/� fibroblasts as a calreticulin

expression system may prove useful to assess functionalities

of calreticulins from different species.

Keywords: Endoplasmic Reticulum — Plant — Arabidopsis

— Tunicamycin — Bradykinin — Calcium.

Abbreviations: BSA, bovine serum albumin; CNX, calnexin;CRT, calreticulin; DTT, dithiothreitol; ER, endoplasmicreticulum; GUS, b-glucuronidase; HA, hemagglutinin; InsP3,inositol 1,4,5-trisphosphate; PBS, phosphate-buffered saline;PEPC, phosphoenolpyruvate carboxylase; PMSF, phenylmethyl-sulfonyl fluoride; PVDF, polyvinylidene difluoride; RT–PCR,reverse transcription–PCR; SHD, SHEPHERD; UPR, unfoldedprotein response.

Introduction

The endoplasmic reticulum (ER) plays a vital role

in a variety of cellular processes including Ca2þ storage

and release, lipid and protein synthesis, protein folding and

post-translational modifications (Trewavas and Malho

1998, Michalak et al. 1999, Baumann and Walz, 2001,

Persson and Harper, 2006). The ubiquitous ER protein

calreticulin (CRT) is important for several of these

processes in animal cells, including Ca2þ homeostasis

(Camacho and Lechleiter 1995, Mery et al. 1996,

Arnaudeau et al. 2002) and protein folding (Peterson

et al. 1995, Culina et al. 2004). In addition, animal CRTs

are suggested to be involved in cell adhesion (Coppolino

et al. 1997), apoptosis (Groenendyk and Michalak 2005)

and regulation of gene expression (Burns et al. 1994,

Dedhar et al. 1994). More specifically, CRT is essential for

cardiac development in mice (Mus musculus), and CRT

deficiency (crt�/�) is embryonic lethal (Nakamura et al.

2001a). Cells derived from CRT-deficient embryos have

impaired Ca2þ homeostasis and compromised protein

folding (Nakamura et al. 2001b). The latter became

apparent through reduced binding of the agonist bradykinin

to the cell surface receptor in CRT-deficient cells. It

appeared that the bradykinin-binding receptor, coupled to

inositol 1,4,5-trisphosphate (InsP3)-dependent Ca2þ release

from the ER, is impaired in the crt�/� cells, suggesting that

CRT affects the folding of the bradykinin receptor

(Nakamura et al. 2001b). Expression of mammalian recom-

binant CRT rescues this phenotype, indicating that the

crt�/� mouse cells provide an excellent tool for exploring

functional properties of CRTs.

Although CRT is well characterized in mammals, the

functional roles of plant CRTs are much less well under-

stood, and have only indirectly been associated with protein

folding (Denecke et al. 1995) and Ca2þ homeostasis

(Persson et al. 2001, Wyatt et al. 2002, Akesson et al.

2005). CRT is expressed in most plant tissues (Crofts

and Denecke 1998), with high abundance in flowers

*Corresponding author: E-mail, [email protected]; Fax, þ49-331-567-898149.

Plant Cell Physiol. 49(6): 912–924 (2008)doi:10.1093/pcp/pcn065, available online at www.pcp.oxfordjournals.org� The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.All rights reserved. For permissions, please email: [email protected]

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(Nelson et al. 1997, Borisjuk et al. 1998). In the plant cell,

CRT is mainly localized to the ER (Opas et al. 1996,

Borisjuk et al. 1998), but has also been shown to be enriched

in the Golgi apparatus in Liriodendron tulipifera, possibly

during maturation of the CRT protein (Navazio et al.

2002), and in the nuclear envelope in maize (Zea mays)

and tobacco (Nicotiana tabacum; Denecke et al. 1995,

Napier et al. 1995). Furthermore, CRT may be unevenly

distributed within the ER, with possible enrichment

associated with plasmodesmata in several tissues (Baluska

et al. 1999, Chen et al. 2005). The apparent complexity in

expression and localization of plant CRTs may be explained

by developmental and tissue-dependent variations, but

may also be due to different homologs of the protein

(Persson et al. 2002, Persson et al. 2003). Two or more

CRT genes are present both in plants and in mammals

(Nelson et al. 1997, Persson et al. 2002, Persson et al. 2003).

Phylogenetic studies and expression analyses of Arabidopsis

(Arabidopsis thaliana) as well as rice (Oryza sativa) have

shown that CRTs in higher plants form two distinct groups;

CRT1a/CRT1b and CRT3 (Persson et al. 2003), suggest-

ing that the observed complexity in CRT expression and

distribution in plants reflect different isoforms of the

protein.

In this study, we focused on functional characterization

of the Arabidopsis CRT isoform AtCRT1a. We used crt�/�

mouse embryonic fibroblasts as the model system to

investigate AtCRT1a functions as a chaperone and Ca2þ

storage protein. Expression of AtCRT1a in crt�/� mouse

cells rescued the mutant phenotypes, which provide the first

direct evidence that the essential functional properties

of CRTs are conserved across the two Kingdoms.

Furthermore, Arabidopsis mutant analyses established

that AtCRT1a has a key role in alleviating the tunicamy-

cin-induced unfolded protein response (UPR) in higher

plants.

Results

Expression of AtCRT1a

From a comparison of the two fully sequenced

genomes of Arabidopsis and rice, it is clear that the

CRT1a/1b and CRT3 groups evolved before the split

between monocots and eudicots (Fig. 1A). To evaluate the

functional properties of specific CRT isoforms, we here

focus on one of the CRTs in the CRT1a/b cluster, CRT1a,

in Arabidopsis. We chose to focus on AtCRT1a because of

its relatively high abundance and because of its rapid

induction in response to tunicamycin, a blocker of N-linked

glycosylation and inducer of the UPR (Persson et al. 2003).

First the relative expression levels were assessed using

semi-quantitative reverse transcription–PCR (RT–PCR)

(Fig. 1B). AtCRT1a transcripts were present in flowers,

siliques, rosette leaves, roots, pedicels and cauline leaves at

similar expression levels (Fig. 1B). This prompted a more

detailed investigation of the expression within the respective

plant organs. We generated transgenic plants where a

b-glucuronidase (GUS) reporter gene was placed under the

control of the 1.5 kb promoter region upstream of the

AtCRT1a open reading frame. In roots, GUS activity was

strongest in the root tips, suggesting a role for CRT in

growing tissues (Fig. 1C). GUS activity was also detected in

floral tissues, specifically associated with the ovules, and

in and around vascular tissues in rosette leaves (Fig. 1C).

A

C

B

Fig. 1 Phylogenetic analysis of CRTprotein sequences in plants and expressionanalysis of AtCRT1a. (A) Phylogeneticanalysis of CRT amino acid sequencesfrom Arabidopsis and rice. Human CRT1was used as the outgroup. (B) Expressionanalysis of AtCRT1a in different tissues inArabidopsis assessed by semi-quantitativeRT–PCR. Upper panel: AtCRT1a (tissuesfrom 4-week-old plants). Amplification ofACTIN RNA was used as control (lowerpanel). (C) Expression of AtCRT1a assessedby a promoter: GUS reporter gene fusionin transgenic Arabidopsis. Arrows indicatemost prominent expression in the root tip of14-day-old seedlings (left panel), ovules infloral tissue (middle panel) and vasculartissue in rosette leaves of 14-day-oldseedlings (right panel).

Functions of calreticulins in higher plants 913

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The expression patterns correlated well with microarray-

based databases, such as Genevestigator (https://www.

genevestigator.ethz.ch; Zimmermann et al. 2004).

Ca2þ binding properties of AtCRT1a

Plant CRTs seem to have a large Ca2þ holding capacity

(Borisjuk et al. 1998, Persson et al. 2001). These results have

mainly been obtained from plant extracts where multiple

CRT isoforms could not be distinguished, and only limited

information is therefore available for individual CRT

isoforms in plants. Analogous to CRT1 from human and

mouse, AtCRT1a has high levels of the negatively charged

aspartic and glutamic acids in the C-terminal region

(Fig. 2A). The negatively charged amino acids in this

region are believed to be responsible for the Ca2þ-holding

capacity in mammalian CRT proteins (Baksh and Michalak

1991). To test whether AtCRT1a may bind Ca2þ in vitro,

we generated a 6�histidine (6�His)-tagged AtCRT1a

construct, expressed it in Escherichia coli and purified the

recombinant protein (Fig. 2B, left panel). Ca2þ binding of

the recombinant AtCRT1a was investigated using 45Ca2þ

overlay of protein blots with a 6�His-tagged human (Homo

sapiens) CRT1, HsCRT1, as positive control. Distinct

radiolabeled bands were detected at apparent molecular

masses in SDS-PAGE of 60 kDa for AtCRT1a and at

56 kDa for HsCRT1 (Fig. 2B, right panel). These bands

corresponded to two protein bands detected with an anti-

6�His antibody by Western blot analysis (data not shown)

and by Coomassie Brilliant Blue staining (compare panels,

Fig. 2B). No labeling was detected for a 6�His-tagged

phosphoenolpyruvate carboxylase (PEPC), used as negative

control, confirming that under these conditions the His tag

itself did not bind Ca2þ (Fig. 2B, right panel). These data

show that both HsCrt1 and AtCrt1a are able to bind Ca2þ

in vitro.

Expression of AtCRT1a in crt�/� mouse embryonic

fibroblasts

Mutant analysis of rabbit (Oryctolagus cuniculus)

CRT1 revealed that several amino acids are essential for

protein folding and Ca2þ storage capacity in animal CRTs

(Martin et al. 2006). These amino acids are conserved in

HsCRT1, mouse (M. musculus) CRT1 and AtCRT1a

(Fig. 2A). In addition, both AtCRT1a and HsCRT1 bind

Ca2þ in vitro (Fig. 2B, right panel), suggesting that the basic

functional CRT properties may be conserved between

plants and animals. To test whether AtCRT1a has similar

function to its animal counterpart, we generated a construct

where an AtCRT1a cDNA was fused to a hemagglutinin

(HA) tag (Fig. 3A) and transformed it into crt�/� mouse

embryonic fibroblasts. Six stable transformants were

isolated using zeocin as a selectable marker, and were

assessed using immunoblotting with anti-HA antibodies.

A typical result is shown in Fig. 3B for two of these

AtCRT1a transgenic crt�/� cell lines (AtCRT1a-3 and -11).

A single 60-kDa protein band was observed in both cell

lines, while no signal was detected in the crt�/� cell line, or

in mock-transfected cell lines, confirming that the observed

protein bands corresponded to the recombinant AtCRT1a.

Immunofluorescence studies of crt�/� cells expressing

AtCRT1a using the anti-HA antibodies revealed a reticu-

lar-like network reminiscent of the ER (Fig. 3C).

To confirm that this network indeed was ER, we also

immunolabeled the cells with antibodies against calnexin

(CNX), an ER-localized chaperone (Fig. 3D). The CNX

pattern of staining in a reticular-like network overlapped

with that of the HA tag antibodies confirming that the

recombinant AtCRT1a was targeted to the ER in the crt�/�

mouse fibroblasts (Fig. 3E).

AtCRT1a restores Ca2þ deficiencies in crt�/� mouse

fibroblasts

The mouse crt�/� fibroblasts have reduced ER Ca2þ

storage capacity (Fig. 4A; Nakamura et al. 2001b).

To assess whether the introduction of the recombinant

AtCRT1a could restore this deficiency, the AtCRT1a-3 and

AtCRT1a-11 cell lines were cultured for 50 h with 45Ca2þ

and then resuspended in a Ca2þ-free buffer. The cells were

pre-incubated at 378C followed by addition of thapsigargin,

an inhibitor of the SERCA pump (Thastrup et al. 1990),

to measure Ca2þ in rapidly exchangeable stores (Nakamura

et al. 2001b), or by addition of the Ca2þ ionophore

ionomycin, to measure the total amount of Ca2þ in the

cell. Released Ca2þ was determined in the supernatant after

pelleting the cells (background values collected in the

absence of thapsigargin or ionomycin accounted for

approximately 40% and are subtracted from data presented

in Fig. 4). Addition of thapsigargin caused a 2-fold increase

in Ca2þ release in AtCRT1a-3 and -11 compared with the

crt�/� and mock-transfected cells (Fig. 4A). The increase

of Ca2þ in thapsigargin-sensitive stores for AtCRT1a-3

and -11 was highly significant as measured by t-tests

(t2¼ 13.7, P¼ 0.000164 for AtCRT1a-3; t2¼ 1.1,

P¼ 0.322455 for AtCRT1a-11) comparing stably trans-

fected clones with the crt�/� fibroblasts. The thapsigargin-

triggered Ca2þ release from the AtCRT1a transgenic lines

was, furthermore, of similar size to those of the wild-type

control line. Addition of ionomycin caused a larger Ca2þ

release compared with thapsigargin (Fig. 4B). The extra

Ca2þ released by ionomycin is most probably emanating

from intracellular compartments other than the ER, such as

the mitochondria. Thus, our data show that AtCRT1a

restored thapsigargin-sensitive Ca2þ stores in the crt�/�

mouse fibroblasts.

914 Functions of calreticulins in higher plants

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https://www

AH. sapiens Crt1M. musculus Crt1A. thalina Crt1a

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M A K L N P K F I S L I L F A L V V I V S A E V I F E E K F E D G - - W E K R W V K S D W K K D D N

H. sapiens Crt1M. musculus Crt1A. thalina Crt1a

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Fig. 2 Comparison of CRT protein sequences and Ca2þ binding of recombinant AtCRT1a. (A) Comparison of amino acid sequences ofhuman CRT1, mouse CRT1 and Arabidopsis CRT1a (GenBank accession Nos. AAA51916, NP_031617 and AAC49695, respectively).Identical and similar amino acids are highlighted in gray. The approximate positions of the histidine-rich N-terminus (N domain), theproline-rich internal region (P domain) and the negatively charged C-terminus (C domain) are indicated with arrows. The putative ER signalsequence (SS) and the ER-retrieval signal (ER-R) are also indicated. The vertical black arrows indicate three highly conserved cysteineresidues. The asterisk indicates a conserved histidine residue in the N domain. Four amino acids (Glu239, Asp241, Glu243 and Trp244,numbering referring to the HsCRT1sequence) that are highlighted in black boxes have been implicated in interactions between ERp57 andanimal CRT1s. (B) Purified recombinant proteins (5 mg per lane) were separated by SDS–PAGE and either stained with Coomassie BrilliantBlue R250 (left panel) or transferred to a PVDF membrane and probed with 45Ca2þ (right panel). His-tagged PEPC was used as a negativecontrol for 45Ca2þ binding. MW, lane with molecular weight standards.


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AtCRT1a restores folding deficiencies in crt�/� mouse

fibroblasts

Addition of the agonist bradykinin causes a rapid Ca2þ

release from the ER via an InsP3-mediated pathway (Hashii

et al. 1993). However, crt�/� cells have impaired bradykinin

cell surface receptors, most probably due to misfolding of

the receptor, and are thus less responsive to bradykinin

(Nakamura et al. 2001b). To investigate if AtCRT1a could

restore the bradykinin-induced Ca2þ release, we treated

cells from the AtCRT1a-3 and -11 cell lines with bradykinin

and measured the Ca2þ release using the fluorescent Ca2þ

indicator Fura 2 (Nakamura et al. 2001b; Fig. 5A, B).

The resting cytoplasmic free Ca2þ concentration was

�60–80 nM for all the cell lines before bradykinin treatment

(Fig. 5A). Addition of bradykinin resulted in a rapid

spike of Ca2þ release (Fig. 5A). The spike was typically

1.5- to 2-fold higher in AtCRT1a-complemented crt�/�

fibroblasts compared with the crt�/� lines and mock-

transfected crt�/� lines (Fig. 5A, B). The difference was

highly significant as evidenced by t-tests (t2¼ 2.4, P¼ 0.09

for AtCRT1a-3; t2¼ 11.3, P¼ 0.001 for AtCRT1a-11).

The Ca2þ release in the complemented cell lines reached

levels comparable with those of wild-type cells (Fig. 5B),

indicating that AtCRT1a restored bradykinin-dependent

Ca2þ release from the ER.

To assess whether the differences in bradykinin-

stimulated Ca2þ release in mutant cell lines were due to

changes in binding of bradykinin to the membrane receptor,

we performed bradykinin binding analyses (according to

Nakamura et al. 2001b). The different cell lines were

incubated with 3H-labeled bradykinin and the amount

of specific binding was then calculated. Both cell lines

B

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N-domain P-domain C-domain HA-tag HDELA

Fig. 3 Expression and localization of AtCRT1a in crt�/� mouse fibroblasts. (A) Schematic view of the HA tag localization in the AtCRT1aconstruct. (B) Western blot analysis of crt�/� mouse fibroblasts transfected with AtCRT1a expression vector. Cells were lysed, and proteinswere separated by SDS–PAGE, transferred to a PVDF membrane and probed with anti-HA tag antibodies. crt�/�, CRT-deficient mousefibroblasts; Empty vector, mock-transfected crt�/� mouse fibroblasts; AtCRT1a-3 and -11, CRT-deficient mouse fibroblasts transfected withAtCRT1a. (C) Immunolabeling of AtCRT1a-3 transgenic crt�/� fibroblasts with anti-HA tag (CRT1a) and (D) with anti-CNX-tag (ER marker)antibodies. (E) Overlay, the yellow color indicates identical localization of the two markers. Scale bar¼ 25 mm.


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expressing AtCRT1a (AtCRT1a-3 and AtCRTa-11) showed

inreased binding of 3H-labeled bradykinin compared with

crt�/�, and for AtCRTa-11 reaching levels similar to the

wild-type cell line (Fig. 5C), indicating that binding abilities

of bradykinin were restored in crt�/� cells expressing

AtCRT1a. Earlier studies demonstrated that the amount

of bradykinin receptors was reduced by 50% in crt�/� lines

(Nakamura et al. 2001b). This fits well with the restoration

of Ca2þ release in complemented cell lines found here,

supporting that AtCRT1a was important for correct folding

of the bradykinin receptor.

AtCRT1a alleviates the tunicamycin-induced unfolded protein

response in Arabidopsis

Expression of AtCRT1a in mouse crt�/� fibroblasts

shows that AtCRT1a is active both in controlling Ca2þ

homeostasis and presumably in protein folding. To assess

its role in planta, we isolated a homozygous Atcrt1a T-DNA

mutant from the SALK collection (SALK_055452;

Fig. 6A). This line showed no detectable transcript levels

(Fig. 6B; lanes 4 and 5), confirming that the T-DNA

insert disrupted expression of the gene. In agreement with

this, one protein band is missing in the microsomal

fraction from the mutant AtCrt1a cell line using maize

CRT antibodies (Fig. 6C; compare lanes 1 and 2). This

antibody recognizes all CRTs as well as a protein band

of an apparent molecular mass of 66 kDa presumably

corresponding to CNX (Persson et al. 2003). It has been

suggested that AtCRT1a harbors more potential N-linked

glycosylation sites compared with AtCRT1b and AtCRT3,

experimentally shown by treatment with N-glycosidase F

(Persson et al. 2003). Thus, AtCRT1a should band at a

higher molecular size than expected based on the number

of amino acids. The disappearance of the CRT band with

higher molecular size in the Atcrt1a background supports

this hypothesis. We conclude that the missing band in

Fig. 6C, lane 2, corresponds to AtCRT1a, and, as predicted

from the number of available N-linked glycosylation

sites, AtCRT1a is the more heavily glycosylated CRT.

No visible effects on growth and morphology were

evident in Atcrt1a plants when grown under normal

conditions (Fig. 7A). Previous studies have shown that

short-term treatment with high concentrations of tunica-

mycin, an inhibitor of N-linked glycosylation and inducer

of the UPR, results in increased CRT expression in

Arabidopsis (Persson et al. 2003), possibly to alleviate the

cell stress. Short-term treatment of 15-day-old plants with

high concentrations of tunicamycin (5 mgml�1 for 6 h) also

resulted in increases at the protein level (Fig. 6D, lane 3).

To test whether longer exposure of tunicamycin sustains the

up-regulation of CRTs, we treated 15-day-old seedlings

with low levels of tunicamycin (0.1 mgml�1) given for the

last 8 d of culture (Fig. 6D, lane 2). The protein levels

corresponding to CRTs were indeed higher compared with

untreated seedlings also with this exposure. The increase in

CRT levels in the seedlings treated for 8 d corresponded to

lower apparent molecular weight bands compared with

control seedlings, as well as to the to 6 h treated seedlings

(compare lane 1 with lanes 2 and 3 in Fig. 6D). With the

prolonged treatment in contrast to the shorter one, all CRT

present had been synthesized during tunicamycin exposure

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crt−/

−

Fig. 4 ER Ca2þ content in crt�/� mouse fibroblasts is restoredby expression of AtCRT1a. Cellular Ca2þ content was determinedby incubation with 55mCi of 45Ca2þ followed by addition ofthapsigargin (A) or ionomycin (B). Wt, wild-type mouse embryonicfibroblasts; crt�/�, CRT-deficient mouse fibroblasts; Empty vector,mock-transfected crt�/� mouse fibroblasts; AtCRT1a-3 and -11,CRT-deficient mouse fibroblasts transfected with AtCRT1a. Thebackground values (counts min�1 in supernatant before additionof thapsigargin or ionomycin) were subtracted. Results representthe average� SE of three independently performed experiments.


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and consequently fewer glycosylated forms are abundant.

Interestingly, a thin band was found of intermediate

apparent molecular weight. This may reflect partly glyco-

sylated CRT1a. No effect on CRT protein or apparent

molecular size was found with 15-day-old seedlings that had

ABK

TG

Time (s)

B

0

50

100

150

200

C

250

300

150

200

50

50 100 150 200 250 300

100

0

0 2000 4000 6000 8000 100000

5

10

15

20

25

[Ca2+

] nM

∆ [C

a2+]

nM

AtC

RT

1a-1

1

AtC

RT

1a-3

Em

pty

vect

or

Wt

crt−/

−

[3 H]-

Bra

dyki

nin-

spec

ific

bin

ding

(fm

ol/m

g pr

otei

n)

[3H]-Bradykinin (pM)

Fig. 5 Bradykinin-induced Ca2þ release in crt�/� mouse fibro-blasts is restored by AtCRT1a. Mouse fibroblasts were loaded withthe fluorescent Ca2þ indicator Fura2-AM followed by stimulationwith bradykinin. (A) Typical traces showing the resting freecytosolic Ca2þ levels before adding the bradykinin (BK) which

induce Ca2þ release from the ER. When the resting level wasrestored, thapsigargin (TG) was added to assess whether the Ca2þ

transport into the ER is functional. After the addition of TG, CaCl2was added to restore the depleted Ca2þ stores. The experimentswere carried out using a Ca2þ-free medium. Filled circles,wild-type cells; filled triangles, crt�/� cells; open triangles, mock-transfected crt�/� cells; open circles (AtCRT1a-3); open squares(AtCRT1a-11), crt�/� mouse cells transfected with AtCRT1a. (B) The�Ca2þ release measured in the presence of bradykinin. Wt, wild-type cells; crt�/�, CRT-deficient cells; Empty vector, mock-transfected crt�/� cells; AtCRT1a-3 and AtCRT1a-11, crt�/� cellstransfected with AtCRT1a. Results represent the average� SEof three independent experiments. (C) Saturation isotherm of[3H]bradykinin binding to wild-type cells (circles), CRT-deficientcells (squares), AtCRT1a-3 (diamonds) and AtCRT1a-11 (triangles)complemented crt�/� mouse embryonic fibroblast cell lines.Specific binding was determined as the difference between thetotal and non-specific binding in the presence of unlabeledbradykinin.

A

B C

crt1a

D

1 2 3 4 1 2 3 45 6

CRTsCNX

1 2 3

CNXCRTs

Fig. 6 Generation and complementation of the Atcrt1a mutantand effect of tunicamycin on CRT protein levels. (A) Relativeposition of T-DNA insertion in the Atcrt1a mutant. Gray boxes,exons; black lines, introns. (B) Analysis of AtCRT1a expressionusing RT–PCR with RNA isolated from wild-type (lanes 1, 2 and 3)and Atcrt1a mutant (lanes 4, 5 and 6) plants, using primersupstream (lanes 1 and 4) and downstream (lanes 2 and 5) of theinsertion site. Amplification of ACTIN mRNA was used as control(lanes 3 and 6). (C, D) Levels of AtCRT protein isolated from15-day-old Arabidopsis seedling cultures were analyzed bySDS–PAGE followed by immunoblot analysis using a maize CRTantibody. (C) Lane 1, wild-type; lane 2, Atcrt1a mutant; lanes 3and 4, Atcrt1a complemented with AtCRT1a under control of a 35Spromoter (two different lines). (D) Wild-type seedling culturestreated with tunicamycin for different times at the end of thegrowth period. Lane 1, no tunicamycin; lane 2, plants treatedwith 0.1 mgml�1 tunicamycin for 8 d; lane 3, plants treated with5mgml�1 tunicamycin for 6 h. A 10 mg aliquot of protein wasloaded per lane.


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been treated with dithiothreitol (DTT), either at a low

concentration (0.2mM) for 8 d (fresh DTT was added every

second day) or at a high concentration (10mM) for 6 h,

supporting that the response to tunicamycin was not a

general stress response, but indeed related to the tunicamy-

cin-induced UPR (data not shown). To test whether the

deletion of AtCRT1a affected the tunicamycin sensitivity,

we treated 1-week old Atcrt1a and wild-type seedlings

with low tunicamycin concentrations (0.1mgml�1) for 8 d.

In agreement with the above, the tunicamycin treatment

induced CRT expression in the treated seedlings compared

with untreated seedlings (data not shown), probably

reflecting expression of CRTs other than AtCRT1a.

After 8 d, the tunicamycin-treated (0.1mgml�1) Atcrt1a

seedlings were smaller compared with wild-type seedlings

(Fig. 7B). To assess which tissues were most affected

in Atcrt1a in response to the tunicamycin treatment, we

measured leaf size, and fresh and dry weights of treated and

control seedlings (Fig. 7C). t-tests revealed that all differ-

ences between values of Atcrt1a mutant seedlings compared

with wild-type seedlings were significant (Fig. 7). To ensure

that the tunicamycin-sensitive phenotype (Fig. 7) was due to

the disruption of the AtCRT1a gene, we complemented

the mutant with an AtCRT1a cDNA under the control

of a 35S promoter (Figs. 6C, lanes 3 and 4). The comple-

mented mutant was indistinguishable from the wild-type

seedlings after tunicamycin treatment (Fig. 7B, C), con-

firming that the drug-sensitive phenotype was indeed due

to disruption of the AtCRT1a gene. Thus, AtCRT1a is

protecting the plant against tunicamycin stress and may

therefore be viewed as an alleviator of the tunicamycin-

induced UPR in Arabidopsis.

Discussion

Plant CRTs are generally assumed to have retained

functions attributed to mammalian homologs. However,

no functional characterization of plant CRTs has been

presented. We show here that the Arabidopsis CRT isoform,

AtCRT1a, can substitute for animal CRTs with regard to

Ca2þ binding and protein folding abilities. Consistent with

this, the amino acid residues that are necessary for these

functions in mammalian CRTs are conserved in AtCRT1a

(Fig. 2A). These amino acid residues are also conserved in

AtCRT1b, suggesting that AtCRT1b may be functionally

Fig. 7 The Atcrt1a mutant is sensitive to tunicamycin treatment.Seeds from wild-type, Atcrt1a, and Atcrt1a complemented withan AtCRT1a cDNA were germinated and grown on normal MSmedium for 1 week, after which they were transferred to the samemedium� tunicamycin and grown for another 8 d. (A) Control,no tunicamycin. (B) Tunicamycin-treated plants. Tunicamycin(0.1 mgml�1) was added for 8 d at the end of the growth period.Data shown in A and B are from one experiment. All experimentshave been repeated at least three times with similar phenotypictraits, and the size of the scale bars is 10mm. Wt, wild-type;Atcrt1a, Atcrt1a mutant; Atcrt1a:AtCRT1a, Atcrt1a mutant com-plemented with AtCRT1a. (C) Leaf size, fresh weight and dry weight

of seedlings exposed to tunicamycin are shown as a percentage,with wild-type levels as 100%. Black bars, wild-type; white bars,Atcrt1a mutant; gray bars, Atcrt1a complemented with AtCRT1a.Results represent the average of three independent experiments.The differences between the tunicamycin-treated wild-type andmutant were significant (t19¼ 4.2; P¼ 0.0001 for leaf size, t9¼ 7.4,P¼ 7.74E-05 for fresh weight and t9¼ 3.4, P¼ 0.009 for dry weightmeasurements).


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redundant with AtCRT1a. Interestingly, the plant CRT1a

protein was found to be located in the ER in the mouse cell,

despite a slightly different retrieval signal (HDEL compared

with KDEL), suggesting that the animal cell sorting

machinery also worked for the plant protein in this case.

Generation of an Atcrt1a mutant only displayed

a conditional phenotype in response to tunicamycin

(Fig. 7), a blocker of N-linked glycosylation and therefore

an inducer of the UPR in the ER (Liu et al. 2007). CRT

deficiencies in other species have resulted in more severe

phenotypes (Nakamura et al. 2001a). For example, CRT

deficiency in mouse causes cardiac arrest and is therefore

embryonic lethal (Nakamura et al. 2001a). We therefore

anticipated a more severe phenotype in CRT-deficient

Arabidopsis plants. The weak phenotype of Atcrt1a may

consequently suggest that other CRT isoforms, most

probably AtCRT1b, may substitute for AtCRT1a in the

Atcrt1a mutant. Should AtCRT1b functionally compensate

for AtCRT1a in the Atcrt1a background we would antici-

pate that the proteins, and possibly genes, are produced and

expressed, respectively, at similar developmental stages

and tissue types. In agreement with this, AtCRT1a and

AtCRT1b are transcriptionally tightly coordinated over

approximately 1,200 microarray data sets (Obayashi et al.

2006, Thelin et al. unpublished). Generation of double

mutants between Atcrt1a and 1b may therefore be infor-

mative for deciphering the effect of removal of CRT activity

in Arabidopsis. The expression pattern of the third CRT

isoform in Arabidopsis, AtCRT3, on the other hand, differs

substantially from that of AtCRT1a and 1b (Persson et al.

2003, Obayashi et al. 2006, Thelin et al. unpublished),

suggesting that AtCRT3 may have a role complementary

to that of the other CRT isoforms. Interestingly, several of

the well conserved amino acids that have been shown to be

essential for basic CRT functions, such as protein folding

and Ca2þ binding abilities, are not conserved in AtCRT3

(data not shown). A comparison using the mouse crt�/�

fibroblasts for AtCRT1a and AtCRT3 may therefore

reveal functional divergence among the CRT isoforms in

Arabidopsis. A more general approach comparing CRT

amino acid sequences from various species may similarly

prove suitable to identify essential amino acids, and may

allow inference of functional roles for specific CRTs.

Several other chaperones, such as CNX1, CNX2 and

SHEPHERD (SHD), are also highly co-expressed with

AtCRT1a and 1b (Obayashi et al. 2006, Thelin et al.

unpublished), indicating that a tightly knit network of

chaperone-like proteins may work together in higher plants.

Considering that CRTs and CNXs may mimic each other

during interactions with maturing proteins (Danilczyk et al.

2000), CNX functions may also partly suppress mutant

phenotypes in Atcrt1a. It may therefore be necessary also

to mutate the CNXs, in addition to the CRTs, to abolish

the ER quality control cycle completely in Arabidopsis.

Mutation in SHD, encoding a GRP94 homolog, causes

enlarged shoot and root apical meristems, and pistil

swelling (Ishiguro et al. 2002). As mentioned above, SHD

is highly co-expressed with AtCRT1a, possibly reflecting

functional relatedness between SHD and AtCRT1a.

Interestingly, AtCRT1a showed very specific expression in

the expansion zone of roots and in floral tissues, specifically

in the ovule (Fig. 1), suggesting that AtCRT1a indeed

may be important for the pistil and root development

similarly to SHD.

CRTs are believed to be the main source of Ca2þ

sequestration in plant cells (Navazio et al. 1995, Persson

and Harper 2006), and directly influence ER Ca2þ levels

in vitro (Persson et al. 2001) and in vivo (Wyatt et al. 2002).

Consistent with this, AtCRT1a restored deficiencies in Ca2þ

homeostasis in crt�/� mouse embryonic fibroblasts (Fig. 4).

As apparent in animals, CRT is a key connector of Ca2þ

signaling and ER protein folding, i.e. the disruption of

CRT in mouse impairs ER Ca2þ release most probably by

misfolding of the bradykinin receptor (Nakamura et al.

2001a). In addition, the ER Ca2þ levels are important for

ER chaperone interactions and therefore protein folding

efficiency in animal cells (Corbett et al. 1999). This implies

that CRT deficiency reduces both ER Ca2þ and the ER

protein folding efficiency. No data linking ER Ca2þ

homeostasis and ER chaperone efficiency are available for

plants. However, the Ca2þ holding ability of AtCRT1a and

the reduced resistance to the UPR, i.e. increased sensitivity

to tunicamycin, in Atcrt1a suggest that the ER Ca2þ

holding potential and the folding efficiency may also be

linked by CRTs in higher plants. Considering that the

amino acid residues necessary for interactions between

CRTs and ERp57, important for disulfide bond rearrange-

ments, are conserved in AtCRT1a, it appears likely that

these Ca2þ-dependent interactions may also be present in

plants. This hypothesis is further corroborated by restora-

tion of the putative folding deficiency in crt�/� mouse

fibroblasts by AtCRT1a complementation (Fig. 5).

Taken together, we show that the two main CRT-

associated functions, the Ca2þ-regulating and chaperone-

like functions, are conserved between plants and animals.

We also show that disruption of one of the CRTs,

AtCRT1a, causes lower stress tolerance to the UPR in

Arabidopsis. Furthermore, we believe that functional

redundancies for a multitude of CRT homologs from

different species may be explored using the mouse crt�/�

fibroblasts as described here.

Materials and Methods

Computational analysis of CRT protein sequences

Protein sequences for human CRT1, mouse CRT1 andArabidopsis CRT1a (accession Nos. AAA51916, NP_031617 and


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AAC49695, respectively) were obtained from the National Centerfor Biotechnology Information (NCBI; www.ncbi.nlm.nih.gov).Alignment was done using ClustalW, MacVector 8.0 software(Oxford Molecular Group plc, UK). A heuristic search usinga maximum parsimony method was performed on the alignedsequences of seven CRT isoforms from Arabidopsis, rice andhuman using the PAUP v.4b10 software (Sinauer Associates,Sunderland, MA, USA), and bootstrap values based on 1,000replicates were used.

Isolation of Arabidopsis RNA and RT–PCR

Total RNA was isolated from various Arabidopsis tissues(4 weeks old) using a Qiagen RNeasy Plant Mini Kit (Qiagen Inc.,Valencia, CA, USA). RNA (1mg) was digested with DNase(Promega, Madison, WI, USA) followed by cDNA synthesiswith an iScript cDNA Synthesis Kit (Bio-Rad, Hercules,CA, USA). Reverse transcription–PCR (RT–PCR) primers forAtCRT1a were as follows: forward primer 50AGCACAAGGATGCGGAGA-30 and reverse primer 50-TCAGAGTCGGATTCAGC-30, and were designed over exon–exon junctions.ACTIN was used as control.

Expression analysis of AtCRT1a

A 1.5 kb long nucleotide sequence upstream of the ATGstart codon comprising the promoter and 50 untranslated regionof AtCRT1a was amplified from genomic Arabidopsis DNA. Theproduct was recombined into the pENTR D-TOPO vector usingthe Gateway system (Invitrogen, Carlsbad, CA, USA) and theninserted into the pKGWFS7 destination vector (Plant SystemsBiology VIB-Ghent University, Belgium) using the LR recombina-tion mix (Invitrogen). The vector was transformed into electro-competent Agrobacterium tumefaciens (strain GV3101) andtransformed into wild-type (col-0) Arabidopsis using the floraldip method (Clough and Bent 1998). Transgenic seeds wereselected on 1/2 Murashige and Skoog (MS) agar plates with50 mgml�1 BASTA (glufosinate ammonium, Fluka, Switzerland),and third-generation seeds were analysed. To visualize GUSexpression patterns, the plant materials were incubated in GUSsubstrate solution [100mM potassium phosphate buffer, pH 7.0,1mM EDTA, 0.1% (w/v) Triton X-100, 12mg ml�1 5-bromo-4chloro-3-indolyl b-D-glucuronic acid (Duchefa Biochemie,Haarlem, The Netherlands)], vacuum infiltrated for 30min,incubated at 378C overnight and destained in 70% (v/v) ethanol.The results shown are representative for three homozygous lineswith similar expression patterns.

Expression and purification of recombinant CRT

For expression of human CRT1 and Arabidopsis CRT1a inE. coli, the full-length CRT genes were amplified and cloned intoa pET15b plasmid (Novagen, Madison, WI, USA). The 6�His-tagged proteins were expressed in BL21 (E. coli) cells in LBmedium containing 100 mg ml�1 ampicillin. Cultures were grown tomid-log phase followed by the induction of expression by 1mMisopropyl-b-D-thiogalactopyranoside (IPTG) for 4 h. Cells werecentrifuged at 4,600� g for 15min and resuspended in 50mMTris–HCl, pH 8.0, 300mM NaCl, 2mM phenylmethylsulfonylfluoride (PMSF) and protease inhibitor cocktail (10 ml ml�1)followed by sonication at 22W for 240 s, and then centrifuged at52,000� g for 20min. The lysates were mixed and incubatedovernight with Ni2þ-nitrilotriacetic acid–agarose beads (QiagenInc.) and 20mM imidazole. After incubation the beads werewashed with 50mM Tris–HCl, pH 8.0, 300mM NaCl and 50mMimidazole, and the His-tagged proteins were eluted with 50mM

Tris–HCl, pH 8.0, 300mM NaCl and 300mM imidazole. Proteindetermination was according to Bearden (1978) using a dye-binding assay with bovine serum albumin (BSA) as standard.

45Ca2þ overlays of protein blots

Purified proteins were separated by SDS–PAGE and trans-ferred to an Immobilon polyvinylidene difluoride (PVDF) transfermembrane (Millipore, Bedford, MA, USA). The membrane waswashed three times for 10min with 60mM KCl, 5mM MgCl2and 10mM imidazole-HCl, pH 6.8, and then incubated overnightwith 60mM KCl, 5mM MgCl2, 10mCi ml�1 45Ca2þ and 10mMimidazole-HCl, pH 6.8 (Maruyama et al. 1984). The membranewas then washed for 10min with deionized water and dried.Radiolabeled proteins were detected with PhosphorImager(GE Healthcare, Buckinghamshire, UK).

Generation of transgenic AtCrt1a mouse embryonic fibroblasts

A full-length AtCRT1a cDNA (GenBank accessionNo. U66343) was cloned into a pcDNA3.1/Zeo vector containingan HA tag in the C-terminus, upstream of the ER retention signal(HDEL) of AtCRT1a to generate pcDNA-CRT1a-HA (Fig. 3A).Wild-type and crt�/� mouse embryonic fibroblasts were grown at378C in a 5% CO2 environment in Dulbecco’s modified Eagle’smedium containing 10% (w/v) fetal bovine serum and 1% (w/v)pencillin streptomycin-glutamine (Nakamura et al. 2001a). crt�/�

cells were transfected with pcDNA CRT1a-HA using EffecteneTransfection Reagent (Qiagen Inc.), and stably transfected celllines were selected in the presence of 400 mg ml�1 zeocin. Six ofthese cell lines were analyzed in more detail with respect toAtCRT1a protein expression, subcellular localization, and effectson Ca2þ homeostasis and protein folding. Typical results shownare from cell lines AtCRT1a-3 and AtCRT1a-11.

SDS–PAGE and immunoblotting

SDS–PAGE was carried out essentially according to Laemmli(1970) with a Bio-Rad Mini-Protean II electrophoresis system.For mouse embryonic fibroblasts, the cells were lysed with RIPAbuffer [50mM Tris–HCl, pH 7.5, 150mM NaCl, 1mM EGTA,1mM EDTA, 1% (w/v) Triton X-100, 0.5% (w/v) deoxycholicacid, 0.1% (w/v) SDS, 1mM benzamidine, 1mM PMSF,0.025mg ml�1 aprotinin, 0.01mg ml�1 pepstatin, 0.05mg ml�1

E-64, 0.025mg ml�1 leupeptin and 0.1mg ml�1 tosyl phenylalanylchloromethyl ketone (TPCK)] and proteins were solubilized byaddition of sample buffer [125mM Tris–HCl, pH 6.8, 4%(w/v) SDS, 20% (v/v) glycerol, 5% b-mercaptoethanol and0.02% bromphenol blue], and separated using a 12% SDS–polyacrylamide gel. For immunoblotting analysis, proteins wereelectrophoretically transferred to a PVDF membrane (Millipore)for 1 h at 100V. After transfer, the membrane was blocked with5% blocking solution [5% (w/v) skimmed milk powder in 10mMTris–HCl, pH 8.0, 150mM NaCl], and washed in 10mMTris–HCl, pH 8.0, 150mM NaCl and 0.05% (w/v) Tween-20.For mouse embryonic fibroblasts, the blotting membranes wereprobed with antibodies against the HA tag (1 : 1,000). The HA tagantibodies were detected with anti-mouse IgG–horseradishperoxidase secondary antibodies (1 : 10,000). For plants, themembrane was probed with maize CRT antibodies (1 : 10,000) toscreen for Atcrt1a mutants together with secondary antibodiesanti-rabbit IgG coupled to horseradish peroxidase (1 : 10,000).The immunodecorated bands were visualized using the enhancedchemiluminescence detection method.


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Immunohistochemistry of mouse embryonic fibroblasts expressingAtCRT1a

For intracellular localization of AtCRT1a in mouse embryo-nic fibroblasts, cells were cultured on coverslips, washed withphosphate-buffered saline (PBS; 5mM potassium phosphatebuffer, pH 7.5, 150mM NaCl), fixed with 4% (w/v) formaldehyde,extracted with 0.1% (w/v) saponin, 2% (w/v) milk powder in PBS,and incubated with HA tag monoclonal antibodies (1 : 50) andCNX polyclonal antibodies (Groenendyk et al. 2006; 1 : 800).The HA tag antibodies were detected with fluorescence AlexaFluor anti-mouse secondary antibodies at �em¼ 680 nm and theCNX antibodies with fluorescence Alexa Fluor anti-rabbitsecondary antibodies at �em¼ 546 nm.

Measurements of ER Ca2þ capacity

Mouse embryonic fibroblasts were cultured for 50 h in thepresence of 55 mCi of 45Ca2þ (Nakamura et al. 2001a). Cells werewashed twice with 5mM EDTA in PBS, and then once with 5mMEDTA in culture medium (without fetal bovine serum), detachedfrom Petri dishes by trypsinization [0.25% (w/v) trypsin and 0.02%(w/v) EDTA in Ca2þ/Mg2þ-free PBS], and finally resuspended inCa2þ-free buffer [143mMNaCl, 6mM KCl, 1mM MgSO4, 20mMHEPES-NaOH, pH 7.4, 0.1% (w/v) glucose and 0.1mg ml�1

sulfinpyrazone]. For 45Ca2þ release experiments, aliquots of5� 106 cells ml�1 were pre-incubated for 3min at 378C followedby treatment with thapsigargin or ionomycin for 4min at 378C.Cells were then pelleted by centrifugation (9,600� g for 3min) andthe radioactivity in the supernatant was measured in a BeckmanLS 7800 scintillation counter. Background values, i.e. radioactivityrecovered in the supernatant in control incubations withoutadditions of thapsigargin or ionomycin, were subtracted prior topresentations in the figures.

Fluorescence Ca2þ measurements of mouse embryonic fibroblasts

Cells were incubated with 2 ml ml�1 Fura 2-AM (Sigma-Aldrich) essentially as described in Nakamura et al. (2001a),washed with PBS, trypsinized [0.25% (w/v) trypsin and 0.02%(w/v) EDTA in Ca2þ/Mg2þ-free PBS], centrifuged, resuspended inCa2þ buffer [143mM NaCl, 6mM KCl, 1mM MgSO4, 20mMHEPES-NaOH, pH 7.4, 0.1% (w/v) glucose, 1mM CaCl2and 0.1mg ml�1 sulfinpyrazone], centrifuged and resuspended inCa2þ-free buffer [143mMNaCl, 6mM KCl, 1mM MgSO4, 20mMHEPES, pH 7.4, 0.1% (w/v) glucose and 0.1mg ml�1 sulfinpyr-azone]. The cells were transferred to a cuvette and 2mM EGTAwas added. Fluorescence was measured at �ex¼ 340 nm. When theresting free cytoplasmic Ca2þ level of the cells (basal Ca2þ level)was reached, the cells were stimulated with 600 nM bradykinin(Fig. 5A, B). Thapsigargin (300 nM), a SERCA (ER Ca2þ-ATPase)inhibitor, was later added to the cells to measure the Ca2þ store inthe ER and to control that no Ca2þ release of bradykinin-inducedcells was due to incomplete Ca2þ storage functions in the ER(Fig. 5A). To measure the store-operated Ca2þ influx, 2mMCaCl2 was added to the cells. Ionomycin (7.5mM) together withCaCl2 (4mM) was added to the cells to obtain the maximum value,and to obtain the minimum value 32mM EGTA, 24mMTris–HCl, pH 7.4 and 0.4% (w/v) Triton X-100 were added tothe cells.

[3H]Bradykinin binding assay

Cells were plated at 2� 104 cells ml�1 in 24-well plates for24 h prior to the binding experiments. The culture medium wasremoved and cells were equilibrated on ice for 10min with bindingbuffer (20mMHEPES-NaOH, pH 7.4, 17mMNaCl, 5.4mMKCl,

0.44mM KH2PO4, 0.63mM CaCl2, 0.21mM MgSO4, 0.34mMNa2HPO4, 110mM N-methylglucamine, 0.1% (w/v) BSA and2mM bacitracin; Yang et al. 1999). Cells were then incubatedin the presence of different concentrations of [3H]bradykinin(0.1–20 nM) in the presence or absence of 10 mM unlabeledbradykinin. After 4 h incubation, cells were washed three timeswith a binding buffer, re-suspended in 250 ml of 100mMNaOH perwell, harvested and counted by scintillation counting. The proteinconcentration was measured by DC Protein Assay Bio-Rad.The amount of specific binding was calculated as the total bindingminus the binding in the presence of 10mM unlabeled bradykinin;binding curves were fitted using a Prism program (GraphPad,San Diego, CA, USA).

Screening of the Atcrt1a mutant

Arabidopsis seeds with a T-DNA insertion in the CRT1a genewere obtained from the SALK collection (SALK_055452; Alonsoet al. 2003). To screen for homozygous plants, DNA was extractedusing the CTAB/chloroform-method (CTAB; 2% N-cetyl-NNN-trimethyl-ammonium bromide, 100mM Tris–HCl, pH 8.0, 20mMEDTA and 1.4M NaCl; Murray and Thompson 1980). A T-DNAleft border primer for the insert 50-CGC CCT GAT AGA CGGTTT TTC-30 was used and primers were made approximately600 bp away from and on each side of the insert position: forward50 CCA CCT TTG CTT AGA GCT CGT CC-30, reverse 50-TTGTAA GAT TTT TGT TTTCCCTG-30. For RNA expression, totalRNA was isolated using a Qiagen RNeasy Plant Mini Kit(Qiagen Inc.). RNA (1 mg) was digested with DNase (Promega)followed by cDNA synthesis with an iScript cDNA Synthesis Kit(Bio-Rad). RT–PCR primers for AtCRT1a were as follows:upstream of the T-DNA insert, forward primer 50-TGGCGAAACTAAACCCTAAA-30, and reverse primer 50-ATCTCTTCTCCCATCCATCCATCTT-30, and downstream of the T-DNAinsert, forward primer 50-AGCACAAGGATGCGGAGA-30 andreverse primer 50-TCAGCGTCGGATTCAGC. Primers weredesigned over exon–exon junctions. ACTIN1 was used as control.For protein expression analyses, seedlings were ground with sandin a homogenization buffer (50mM MOPS-KOH, pH 7.5, 0.33Msucrose, 5mM EDTA) with 2.5ml of buffer per g of plant material.The samples were centrifuged for 15min at 5,000�g and thesupernatants were collected for immunoblot analyses.

Complementation of Atcrt1a

A full-length AtCRT1a cDNA (GenBank accession No.U66343) was cloned into a p35SBARN binary vector (Cloughand Bent 1998). The construct was transformed into electrocom-petent A. tumefaciens GV3101, and transformed into the Atcrt1amutant by floral dip (Clough and Bent 1998). Transformed plantswere grown on 1/2 MS medium with 0.8% agar containing25mg ml�1 BASTA (resistance conferred from the AtCRT1atransgene).

Tunicamycin treatment of Arabidopsis seedlings

Growth experiment. Arabidopsis plants were germinated andgrown for 1 week on 1/2 MS medium with 0.8% (w/v) agar at 238Cwith 16 h of light (100mEm�2 s�1), then transferred to 1/2 MSmedium with 0.8% agar� 0.1mg ml�1 tunicamycin and grownfor another 8 d. Control plants (15 d old; no tunicamycin) andtunicamycin-treated plants were photographed, and leaf length,fresh weight and dry weight were measured.

Western blotting experiment. Arabidopsis seedling cultureswere germinated and grown in 250ml flasks [100ml per flaskof Gamborg B5 salt, 1.5% (w/v) sucrose and 0.5mg ml�1


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MES-KOH, pH 5.7]. The cultures were kept on a rotary shaker(125 rpm) for 15 d at 238C, with 16 h of light. At the end ofthe growth period, the medium was supplemented with either0.1 mg ml�1 tunicamycin for 8 d or 5mg ml�1 tunicamycin for 6 h.Seedlings (50mg) from the different treatments and untreatedcontrol were homogenized in 2� SDS–PAGE loading bufferwith 1 ml of protease inhibitor cocktail per 100 ml of buffer. Thesamples were then centrifuged for 2min at 3,000 rpm and 10 mlof the sample supernatants were subjected to SDS–PAGE andimmunoblot analysis.

Statistics

The statistical significance of differences between treatmentsor measurements of different cell lines was assessed by Student’st-test (Microsoft Office Excel, Microsoft Corporation, Redmond,WA, USA). The number of replicates and the level of significanceis given in the text or figure legends.

Funding

The Swedish Research Council (to M.S.); Swedish

Foundation for Strategic Research (to M.S.), The Swedish

Research Council Formas (to M.S.); Carl Tryggers

Foundation (to S.W.); Canadian Institutes of Health

Research (Grant MOP-1529 to M.M.); Alberta Heritage

Foundation for Medical Research (M.M.). J.J. is a recipient

of a studentship award from the Alberta Heritage

Foundation for Medical Research.

Acknowledgments

We are grateful to Monika Dabrowska for superb technicalhelp, Dr. Magnus Alsterfjord for help with the AtCRT1a promoter:GUSconstruct, Lisa Thelin for help with the phylogenetic tree, andDr. Peter Olsson for the gift of the recombinant PEPC protein.

References

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(Received January 23, 2008; Accepted April 15, 2008)


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Functional Characterization of - Plant and Cell Physiology

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