AtIRE1A/AtIRE1B and AGB1 independently control two essential unfolded protein response pathways in...

AtIRE1A/AtIRE1B and AGB1 independently control twoessential unfolded protein response pathways in Arabidopsis

Yani Chen and Federica Brandizzi*

Michigan State University/Department of Energy Plant Research Laboratory and Department of Plant Biology, Michigan State

University, East Lansing, MI 48824, USA

Received 2 August 2011; revised 6 September 2011; accepted 8 September 2011; published online 21 October 2011.

*For correspondence (fax: +1 517 353 9168; e-mail [email protected]).

SUMMARY

The endoplasmic reticulum (ER) has the ability to maintain the balance between demand for and synthesis of

secretory proteins. To ensure protein-folding homeostasis in the ER, cells invoke signaling pathways known as

the unfolded protein response (UPR). To initiate UPR, yeasts largely rely on a conserved sensor, IRE1. In

metazoans, there are at least three independent UPR signalling pathways. Some UPR transducers have been

identified in plants, but no genetic interaction among them has yet been examined. The Arabidopsis genome

encodes two IRE1 sequence homologs, AtIRE1A and AtIRE1B. Here we provide evidence that AtIRE1A and

AtIRE1B have overlapping functions that are essential for the plant UPR. A double mutant of AtIRE1A and

AtIRE1B, atire1a atire1b, showed reduced ER stress tolerance and a compromised UPR activation phenotype.

We have also established that Arabidopsis AGB1, a subunit of the ubiquitous heterotrimeric GTP-binding

protein family, and AtIRE1A/AtIRE1B independently control two essential plant UPR pathways. By

demonstrating that atire1a atire1b has a short root phenotype that is enhanced by an agb1 loss-

of-function mutation, we have identified a role for UPR transducers in organ growth regulation.

Keywords: unfolded protein response, inositol-requiring enzyme 1, GTP-binding protein b1, endoplasmic

reticulum stress, root, Arabidopsis thaliana.

INTRODUCTION

Environmental or physiological factors that cause an accu-

mulation of unfolded proteins in the endoplasmic reticulum

(ER) lead to ER stress. To restore ER protein-folding

homeostasis, eukaryotic cells invoke protective signaling

pathways known as the unfolded protein response (UPR)

(Kozutsumi et al., 1988; Schroder and Kaufman, 2005). The

regulatory mechanisms of UPR have been extensively

explored in yeasts and metazoans. Inositol-requiring

enzyme 1 (IRE1) is a highly conserved protein in eukaryotes

and the major UPR sensor in yeasts (Cox et al., 1993; Mori

et al., 1993). IRE1 is a type I membrane protein that consists

of a UPR sensor domain at the N-terminus as well as kinase

and endoribonuclease domains at the C-terminus. Upon

sensing accumulation of unfolded proteins in the ER, the

endoribonuclease domain of IRE1 is activated by oligomer-

ization and trans-autophosphorylation. Activated IRE1 spli-

ces the transcript of a specific transcription factor – HAC1 in

yeasts (Cox and Walter, 1996) and XBP1 in animals (Shen

et al., 2001). The spliced transcription factors control the

expression of UPR target genes, which are involved in

assisting protein folding, degrading mis-folded protein, and

regulating programmed cell death (Acosta-Alvear et al.,

2007; Cox and Walter, 1996). To manage the UPR induced by

various physiological or environmental conditions, animal

cells use the IRE1 pathway and two additional UPR regula-

tory pathways: the PKR-like ER kinase (PERK) pathway and

the activating transcription factor 6 (ATF6)-dependent

pathway (Ron and Walter, 2007). Similar to IRE1, both PERK

and ATF6 have a UPR sensor domain that protrudes into the

ER lumen, and a cytosolic region that initiates downstream

responses. PERK mediates the UPR by repression of protein

synthesis through phosphorylation of eukaryotic initiation

factor 2a (Harding et al., 2000). ATF6 is a membrane-tethered

transcription factor that is activated by ER stress. The tran-

scriptional activation domain of ATF6 is released from the

ER membrane by protease cleavage and translocated into

the nucleus to regulate UPR genes upon ER stress (Haze

et al., 1999).

A few conserved UPR transducers have been identified in

plants. In Arabidopsis, bZIP28 is known to be a functional

homolog of the mammalian ATF6. To activate the UPR,

bZIP28 undergoes proteolytic release of its transcriptional

266 ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd

The Plant Journal (2012) 69, 266–277 doi: 10.1111/j.1365-313X.2011.04788.x

activation domain to regulate expression of ER chaperones

(Gao et al., 2008; Liu et al., 2007). The Arabidopsis genome

also encodes two sequence homologs of IRE1 – AtIRE1A

(At2g17520) and AtIRE1B (At5g24360) (Koizumi et al., 2001) –

that are ubiquitously expressed in plant tissues (Noh et al.,

2002). Analyses of three individual T-DNA insertion lines of

AtIRE1A showed that induction of UPR genes was similar

between the atire1a mutants and wild-type upon ER stress

(Lu and Christopher, 2008). Recently, it was demonstrated

that UPR gene induction is reduced in a T-DNA insertion line

of AtIRE1B (atire1b-2) (Deng et al., 2011). In addition,

activation of bZIP60 upon ER stress relies on its splicing by

AtIRE1B, but not by AtIRE1A, suggesting that AtIRE1B is the

only functional IRE1 homolog in plants (Deng et al., 2011).

However, the possibility that AtIRE1A also plays a role in the

plant UPR cannot be excluded, as the functional redundancy

between AtIRE1A and AtIRE1B has not yet been tested.

In addition to the conserved counterparts of mammalian

UPR regulators, unexpected UPR mediators have also been

identified in plants. It has been shown that GTP-binding

protein b1 (AGB1), an ER-localized heterotrimeric GTP-

binding protein (G protein), is involved in the UPR in

Arabidopsis (Wang et al., 2007). G proteins are ubiquitous

signaling molecules in eukaryotes (Neer and Clapham,

1988). In plants, AGB1 is known to be involved in vegetative

and reproductive development as well as in light and

oxidative stress responses (Jones et al., 2003; Joo et al.,

2005; Lease et al., 2001; Wei et al., 2008).

In this study, we have performed functional characteriza-

tion of an atire1a atire1b double mutant, providing genetic

and molecular evidence showing that AtIRE1A and AtIRE1B

are essential plant UPR regulators. We also show that an

agb1 loss-of-function mutant enhanced the atire1a atire1b

phenotype with respect to UPR activation. Thus, we have

established that AtIRE1 and AGB1 independently control

two essential plant UPR pathways. The negative genetic

interaction between AtIRE1A/AtIRE1B and AGB1 was further

confirmed by showing the short-root phenotype is aggra-

vated in the atire1a atire1b agb1 triple mutant compared to

the atire1a atire1b double mutant. Hence, this study also

sheds light on the regulation of organ-specific growth by

UPR transducers in plants.

RESULTS

AtIRE1A and AtIRE1B are essential for the plant UPR

To determine whether AtIRE1A and AtIRE1B control the plant

UPR, we performed functional analyses by isolating loss-

of-function mutations of AtIRE1A and AtIRE1B. The atire1a-4

(WISCDSLOX420D09) and atire1b-2 (SAIL_238_F07) alleles

were obtained from the Arabidopsis Biological Resource

Center (Figure 1). Homozygous lines of atire1a-4 and atire1b-

3 were confirmed by genomic PCR (Figure S1). To determine

whether atire1a-4 and atire1b-2 represent RNA null alleles, 4

specific pairs of primers (AtIRE1A-N, AtIRE1A-C, AtIRE1B-N,

and AtIRE1B-C) (Table S1), annealing upstream or down-

stream of the T-DNA insertion sites, were used in RT-PCR

analyses (Figure 1b). We detected no AtIRE1A transcript in

atire1a-4 using either primer set, suggesting that atire1a-4 is

a knockout mutant. In contrast, the AtIRE1B amplicon was

found to be present in atire1b-2 using the upstream primer

set (AtIRE1B-N), but no AtIRE1B amplicon was detectable in

atire1b-2 using the downstream primer set (AtIRE1B-C). The

RT-PCR results indicate that atire1b-2 is not an RNA null

mutant, but it does not express the full-length AtIRE1B

transcript.

To test ER stress tolerance, atire1a-4, atire1b-2 and wild-

type were germinated on medium containing DMSO (mock

control) or 25 or 50 ng/ml tunicamycin (Tm), which is a

typical UPR inducer that blocks protein N-glycosylation. As

atire1a-4, atire1b-2 and wild-type displayed similar

responses with respect to both ER stress tolerance and

UPR gene induction (Figures 2a and S2), we hypothesized

that the two AtIRE1 isoforms could compensate for each

other in the single mutants. To test this possibility, we

generated an atire1a atire1b double mutant by crossing

40 cycles

40 cycles

40 cycles

40 cycles

35 cycles

25 cycles

AtIRE1A-N

AtIRE1A-C

AtIRE1B-N

AtIRE1B-C

AGB1

UBQ10

AtIRE1A

AtIRE1B

AGB1

ire1a-4(a)

(b)

100 bp

ire1b-2 ire1b-1

agb1-2

AtIRE1A-N

AtIRE1B-N

AGB1

agb1-1 (G->A) agb1-3

AtIRE1A-C

AtIRE1B-C

ire1b-3

Figure 1. Isolation of mutants of AtIRE1A, AtIRE1B and AGB1. (a) Genomic

structure of AtIRE1A, AtIRE1B and AGB1. The coding regions and UTRs are

indicated by black and gray rectangles, respectively. The T-DNA insertions in

atire1a-4, atire1b-1, atire1b-2, atire1b-3, agb1-2 and agb1-3 are indicated by

open triangles; the point mutation in agb1-1 is indicated by an arrow.

(b) RT-PCR analyses of AtIRE1A, AtIRE1B, AGB1 and UBQ10 transcript in wild-

type, atire1a-4, atire1b-2 atire1a atire1b, agb1-3 and atire1a atire1b agb1. The

primer locations are shown in (a), and primer sequences are given in Table S1.

Control of the plant UPR by AtIRE1 and AGB1 267

ª 2011 The AuthorsThe Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 266–277

atire1a-4 and atire1b-2 (Figures 1b and S1). The atire1a

atire1b double mutant was over-sensitive to Tm compared

with the wild-type and single mutants (Figure 2a). RT-PCR

analyses showed that, after 6 h Tm treatment, induction of a

known UPR activation indicator, BiP3 (Iwata and Koizumi

2005), was drastically reduced in the atire1a atire1b double

mutant compared to wild-type (Figure 2b). Quantitative real-

time RT-PCR confirmed that the expression level of BiP3 in

the atire1a atire1b double mutant was three to four times

lower than that of wild-type over a 3-day time course of Tm

treatment (Figure 2). Consistently, the induction of two other

UPR genes, AtERdj3A and AtERdj3B (Yamamoto et al.,

2008), was lower in the atire1a atire1b double mutant than

in wild-type upon ER stress induction (Figure 2d). Thus, we

concluded that the lower ER stress tolerance phenotype in

the atire1a atire1b double mutant arose from defects in UPR

gene induction.

To prove that Tm-sensitive phenotype was due to loss of

function of AtIRE1A and AtIRE1B, we complemented the

atire1a atire1b double mutant using AtIRE1A under the

control of the native promoter (pAtIRE1A-AtIRE1A) as well as

using a dexamethasone-inducible clone of either AtIRE1A or

AtIRE1B. We found that AtIRE1A or AtIRE1B alone at least

partially rescued the ER stress-sensitive phenotype of the

atire1a atire1b double mutant (Figure S3). These data show

that the Tm-sensitive phenotype of the atire1a atire1b

double mutant is caused by loss-of-function mutations in

AtIRE1A and AtIRE1B. Therefore, IRE1 signaling is essential

for the plant UPR, and AtIRE1A and AtIRE1B share partially

overlapping functions in the plant UPR activation.

Loss of function of AGB1 causes sensitivity to ER stress

Our data indicate that AtIRE1A and AtIRE1B are essential

for the plant UPR. We further investigated the plant UPR

A

B

D

C

A

B

D

C

DMSO 50 ng ml–1 Tm25 ng ml–1 Tm(a)

A

B

D

C

WT atire1a atire1b

Rel

ativ

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pres

sion

WT atire1a atire1b

BiP3

WT atire1a atire1b

60

40

20

0

2

Rel

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R

elat

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expr

essi

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(h)

BiP3

0 3 6 0 3 6

UBQ10

(b)

(c)

(d1)

1 2 3 (days)

2

1

0

4

01 2 3 (days)

AtERdj3B

AtERdj3A

(d2)

1 2 3 (days)

Figure 2. AtIRE1A and AtIRE1B are required for the plant UPR.

(a) Wild-type (A), atire1a-4 (B), atire1b-2 (C) and atire1a atire1b (D) were germinated on LS medium containing DMSO, 25 or 50 ng/ml Tm for 2 weeks.

(b) RT-PCR of BiP3 transcripts in 4-week-old wild-type and atire1a atire1b after 0, 3 or 6 h treatment with 10 lg/ml Tm in a hydroponic system.

(c) Quantitative real-time RT-PCR of BiP3 transcripts in 2-week-old wild-type and atire1a atire1b after 1, 2 or 3 days treatment with 50 lg/ml Tm in a plate system.

(d) Quantitative real-time RT-PCR of AtERdj3A and AtERdj3B transcripts under the same conditions as (c).

268 Yani Chen and Federica Brandizzi


signaling networks by determining whether another regu-

latory pathway operates in parallel with the AtIRE1A/AtIR-

E1B-dependent signaling pathway. Therefore, we first

compared the ER stress tolerance between mutants of

known UPR components, including bZIP28, bZIP60, BiP2

and AGB1 (Lu and Christopher, 2008; Liu et al., 2007; Wang

et al., 2005, 2007). Among the bzip28-1, bzip60-1, bip2 and

agb1-3 mutants, only agb1-3 showed a Tm-sensitive phe-

notype. We found that the agb1-3 mutant was more sen-

sitive to ER stress compared to wild-type (Figure 3a).

However, another T-DNA allele of AGB1, the agb1-2 mu-

tant, was previously shown to be more resistant to ER

stress (Wang et al., 2007). To clarify whether different agb1

allelic mutants have similar ER stress sensitivity, we

compared the ER stress tolerance between agb1-3 and two

other null alleles of AGB1, agb1-1 and agb1-2 (Figure 1a)

(Lease et al., 2001; Wang et al., 2007) by germinating the

seeds on Tm-containing medium. Similar to the agb1-3

mutant, the agb1-1 and agb1-2 mutants displayed over-

sensitivity to ER stress compared with wild-type (Fig-

ure 3b). Hence, we conclude that there is no substantial

difference in ER stress tolerance among the agb1-1, agb1-2

and agb1-3 mutants. To clarify that the opposite ER stress

tolerance phenotypes of the agb1 mutants reported here

and by Wang et al. (2007) was not due to use of different

phenotypic assays, the higher ER stress sensitivity of agb1-

3 compared to wild-type was further confirmed using the

same ER stress phenotypic assay as used by Wang et al.

(2007). agb 1-3 and wild-type seeds were germinated on

high concentrations of Tm (300 ng/ml) for 6 days, and then

transferred to normal growth medium without Tm for

10 days. Wild-type seeds recovered from the ER stress as

indicated by germination, but the agb1-3 seeds failed to

survive under the same treatment (Figure 3c). In addition,

complementation of agb1-3 by AGB1 showed that the ER

stress phenotype of agb1-3 is due to the AGB1 loss-of-

function mutation (Figure 3d). Therefore, these data pro-

vide evidence that an AGB1 loss-of-function mutation

causes over-sensitivity to ER stress.

AtIRE1A/AtIRE1B and AGB1 independently control two

essential plant UPR sub-pathways

To our knowledge, genetic interaction between plant UPR

transducers has not yet been reported. We have shown that

both AtIRE1A/AtIRE1B and AGB1 are essential for the plant

UPR. We further investigated whether AtIRE1A/AtIRE1B and

AGB1 function in a linear UPR pathway by generating an

atire1a atire1b agb1 triple mutant. Interestingly, compared

to the atire1a atire1b double mutant and the agb1-3 single

mutant, the triple mutant displayed an even more sensitive

phenotype to ER stress using two phenotypic assays: Tm

infiltration and germination on Tm-containing medium

(Figure 4a,b). Leaf senescence and damage were more se-

vere in the atire1a atire1b agb1 triple mutant than in the

atire1a atire1b double mutant at 2–4 days after infiltration

with 15 lg/ml Tm (Figure 4a). When germinated on Tm-

containing medium, the atire1a atire1b agb1 triple mutants

were smaller than the atire1a atire1b double mutants (Fig-

ure 4b). The enhanced growth defects of the atire1a atire1b

agb1 triple mutant compared to the atire1a atire1b double

mutant upon ER stress were further visualized by induced

hypocotyl elongation under dark growth conditions

(Figure 4b).

We investigated whether the lower ER stress tolerance

in the atire1a atire1b agb1 triple mutant compared to the

atire1a atire1b double mutant was the result of more

extensive aberrant UPR gene induction. We compared the

expression of the UPR genes BiP3, AtERdj3A and AtERdj3B

in the agb1-3 single mutant, the atire1a atire1b double

mutant and the atire1a atire1b agb1 triple mutant over the

time course of Tm treatment. The quantitative real-time

RT-PCR results showed that the three UPR genes were

induced to a lower level in the atire1a atire1b double

mutant compared to wild-type, but induction was higher in

the agb1-3 single mutant compared to wild-type (Fig-

ure 4c,d). These data suggest that AtIRE1A/AtIRE1B and

AGB1 play antagonistic roles in UPR gene induction.

Furthermore, although the levels of UPR gene induction

were altered in the atire1a atire1b double mutant and the

agb1-3 single mutant, the expression patterns of UPR

genes in the atire1a atire1b double mutant and the agb1-3

single mutant were similar to those of wild-type over the

time course of Tm treatment (Figure 4d). In contrast, both

the expression levels and patterns of UPR gene expression

were severely affected in the atire1a atire1b agb1 triple

mutant (Figure 4d). Together with evidence that the ER

stress tolerance of the atire1a atire1b agb1 triple mutant

was lower than that of the atire1a atire1b double mutant

and the agb1-3 single mutant (Figure 4a,b), these results

enable us to conclude that AtIRE1A/AtIRE1B and AGB1

mediate two essential plant UPR signaling arms that

operate in parallel. When the two pathways dependent

on AtIRE1A/AtIRE1B or AGB1 are compromised, plants have

a markedly reduced ability to re-program the transcrip-

tional machinery to deal with ER stress. In metazoans,

regulatory relationships have been established between

components belonging to independent UPR pathways. For

example, although ATF6 and IRE1 are known to be two

distinct UPR sensors in metazoans, ATF6 activates the

transcription of XBP1, whose product is the splicing

substrate of IRE1 and a key regulator of the IRE1-dependent

signaling arm (Yoshida et al., 2001). Because transcription

of AGB1 is down-regulated upon UPR activation (Wang

et al., 2007), we investigated whether the AGB1 transcript

is regulated by AtIRE1A/AtIRE1B. Hence, we compared the

AGB1 expression level between wild-type and the atire1a

atire1b double mutant over a time course of Tm treatment.

Consistent with earlier findings (Wang et al., 2007), the



(b)

agb1-3

WT

agb1-3

WT

(d)

agb1-3/35S-YFP-AGB1#3




DMSO 25 ng ml–1 Tm

(a)

b

WT

bzip28-1

bzip60-1

bip2

atire1aatire1b

agb1-3

DMSO

25 50

Tm (ng ml–1)

agb1-3

agb1-1

agb1-2

atire1aatire1b

WT

DMSO

12.5 25

Tm (ng ml–1)

(c)WT atire1a atire1b

agb1-3 atire1a atire1b agb1

atire1a atire1b


WT

Mock Recover from 300 (ng m–1) Tm

Figure 3. Loss of function of AGB1 leads to over-sensitivity to ER stress.

(a) Wild-type, bzip28-1, bzip60-1, bip2, agb1-3 and atire1a atire1b were germinated on LS medium containing DMSO, 25 or 50 ng/ml Tm for 2 weeks.

(b) Wild-type, agb1-1, agb1-2, agb1-3 and atire1a atire1b were germinated on LS medium containing DMSO, 12.5 or 25 ng/ml Tm for 2 weeks.

(c) Wild-type, agb1-3, atire1a atire1b and atire1a atire1b agb1 were germinated on half-strength LS medium containing 300 ng/ml Tm for 6 days, and then transferred

to half-strength LS medium without Tm. The plants were photographed after a 10-day recovery on half-strength LS medium without Tm. For the mock control, the

plants were germinated on half-strength LS medium containing DMSO for 6 days, and then transferred to half-strength LS medium without DMSO or Tm.

(d) Complementation of agb1-3 by 35S–YFP–AGB1. Seeds of wild-type, agb1-3 and agb1-3 expressing 35S–YFP–AGB1 (lines 3 and 8) were germinated on half-

strength LS medium containing DMSO or 50 ng/ml Tm for 2 weeks. Lines 3 and 8 are two independent T2-segregating lines.



AGB1 RNA level decreased upon UPR activation in wild-

type (Figure 4e). However, in the atire1a atire1b double

mutant, the level of AGB1 transcript remained unchanged

over the time course of Tm treatment (Figure 4e). These

results imply that down-regulation of the AGB1 transcript

upon ER stress relies on AtIRE1A/AtIRE1B.

DMSO

Tm 4 day

Tm 3 day

atire1aatire1b

agb1-3 atire1aatire1bagb1

WT

Tm 2 day

25 n

g m

l–1

Tm

25 n

g m

l–1

Tm

(D

ark)

DM

SO

C

D

A

B

C

D

A

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AtERdj3B

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atire1a atire1b agb1R

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AGB1

Rel

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BiP3

WT atire1a atire1b


Rel

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1.5

1

0

agb1-3

AtERdj3A

0.5

0

10

20

30

40

0

1

2

3

4

5

0

5

10

15

1 2 3 4 5 (days) 4 8 24 48 72 (h)

4 8 24 48 72 (h)4 8 24 48 72 (h)

(a) (b)

(c) (d1)

(d2)(e)

Figure 4. agb1-3 enhances the Tm-sensitive phenotype in atire1a atire1b.

(a) Leaves of 5-week-old wild-type, agb1-3, atire1a atire1b, and atire1a atire1b agb1 were infiltrated with DMSO or 15 lg/ml Tm.

(b) Wild-type (A), agb1-3 (B), atire1a atire1b (C) and atire1a atire1b agb1 (D) were germinated on LS medium containing DMSO or 25 ng/ml Tm under either normal

growth or dark conditions for 10 days.

(c) Quantitative real-time RT-PCR of BiP3 transcripts in 2-week-old wild-type, agb1-3, atire1a atire1b and atire1a atire1b agb1 after treatment with 50 lg/ml Tm for 4,

8, 24, 48 or 72 h in a plate system.

(d) Quantitative real-time RT-PCR of AtERdj3A (d1) and AtERdj3B (d2) transcripts in 2-week-old wild-type, agb1-3, atire1a atire1b and atire1a atire1b agb1 after

treatment with 50 lg/ml Tm for 4, 8, 24, 48 or 72 h in a plate system.

(e) Quantitative real-time RT-PCR of AGB1 transcripts in 2-week-old wild-type and atire1a atire1b after treatment with 50 lg/ml Tm for 1, 2, 3, 4 or 5 days in a plate

system.



AtIRE1A and AtIRE1B play a role in root growth

In multicellular organisms, the demand for secretory protein

varies during cell differentiation and proliferation, and UPR

is required for maintenance of the ER protein-folding capa-

bility in specialized cell types or at specific developmental

stages in metazoans. For example, deletion of mammalian

IRE1a causes embryo lethality due to placental defects

(Iwawaki et al., 2009). To investigate whether AtIRE1A/AtIR-

E1B-mediated UPR is involved in growth and development

in plants, we compared plant morphology between wild-

type and the atire1a atire1b double mutant through various

developmental stages. We found that the primary root of the

atire1a atire1b double mutant was significantly shorter than

that of wild-type (t test, P = 3.92449E-18) (Figure 5a,b), but

there was no visible morphological phenotype in the aerial

parts (Figure 5c,d). These results indicate that AtIRE1A and

AtIRE1B are specifically involved in optimal root growth in

plants. In addition, consistent with previous findings (Mud-

gil et al., 2009; Pandey et al., 2008), the agb1-3 single mutant

had longer roots compared to the wild-type (t test,

P = 0.0064) (Figure 5a,b). However, the primary root of the

atire1a atire1b agb1 triple mutant was significantly shorter

than that of the atire1a atire1b double mutant (t test,

P = 0.0024) (Figure 5a,b). The fact that the agb1-3 mutation

enhanced both ER stress sensitivity (Figure 4a,b) and root

growth defects of the atire1a atire1b double mutant sug-

gests that AtIRE1A/AtIRE1B and AGB1 independently regu-

late two parallel UPR pathways, and that both these UPR

pathways contribute to root growth.

The root growth phenotype of atire1a atire1b is

associated with defects in cell elongation

To further explore the short-root phenotype, we visualized

the root tissue anatomy by counterstaining cell walls with

propidium iodide (Figure 6a). Four well-characterized

growth zones are defined in the Arabidopsis root apex: the

meristematic zone, transition zone, elongation zone and

growth-terminating zone (Verbelen et al., 2006). The length

of each zone in Arabidopsis thaliana ecotype Col-0 has been

determined based on their unique cellular activities: meri-

stem, 200 lm from the root cap junction (RCJ); transition

zone, 200–520 lm from the RCJ; elongation zone, 520–

850 lm from the RCJ; growth-terminating zone, 850–

1500 lm from the RCJ (Verbelen et al., 2006). In addition, the

onset of fast elongation in the elongation zone is indicated

by root hair initiation in epidermal cells. The results of pro-

pidium iodide staining show that the cell number and cell

size within 400 lm from the RCJ were similar between wild-

type, the agb1-3 single mutant, the atire1a atire1b double

mutant and atire1a atire1b agb1 triple mutant (Figures 6a

and S4), suggesting an absence of significant defects in

distal root patterning. In sharp contrast, in the region beyond

400 lm, the cell length was abnormal in the atire1a atire1b

double mutant and the atire1a atire1b agb1 triple mutant. In

wild-type and the agb1-3 single mutant, the length of cells

gradually increased from the RCJ towards the growth-ter-

minating zone (Figure 6a). However, the pattern of cell

elongation was different in the atire1a atire1b double mutant

and atire1a atire1b agb1 triple mutant (Figure 6a). Cells

WT (a) (b)

(c) (d)

atire1a atire1b agb1-3 atire1a atire1b agb1

WT atire1a atire1b agb1-3 atire1a atire1b agb1

WT atire1a atire1b


FW (m

g)/p

lant

2

1

0

3

4Leaf FW

0

5

10

WT atire1a atire1b


* *

Primary root length

Roo

t len

gth

(cm

)

* *

Figure 5. agb1-3 enhances the short-root phenotype in atire1a atire1b.

(a) Wild-type, agb1-3, atire1a atire1b and atire1a atire1b agb1 were grown on LS medium for 2 weeks.

(b) Measurement of primary root length (cm) of wild-type, agb1-3, atire1a atire1b and atire1a atire1b agb1. A single asterisk indicates a significant difference

between agb1-3 and wild-type or between atire1a atire1b and wild-type. The double asterisk indicates a significant difference between atire1a atire1b and atire1a

atire1b agb1.

(c) Macromorphology of wild-type, agb1-3, atire1a atire1b and atire1a atire1b agb1.

(d) Fresh weight (mg) of leaves from rosettes of 2-week-old plants.



between 400–600 lm from the RCJ were significantly longer

in the atire1a atire1b double mutant and atire1a atire1b agb1

triple mutant compared to that of wild-type (Figure 6a,b). In

addition, in the elongation zone, the mean cell length of cells

showing root hair initiation was only 50% in the atire1a

atire1b double mutant and 40% in the atire1a atire1b agb1

triple mutant compared to wild-type (Figure 6a,c). These

data indicate that the short-root phenotype of the atire1a

atire1b double mutant and the atire1a atire1b agb1 triple

mutant is due to a disorder in cell elongation in transition

zone/elongation zone rather than defects in the meristem.

The data further imply that maintenance of optimal root cell

elongation relies on the AtIRE1A/AtIRE1B- and AGB1-

dependent signaling pathways.

The expression of UPR genes is lower in the root of the

atire1a atire1b agb1 triple mutant

Our results from propidium iodide staining show that the

atire1a atire1b double mutant and the atire1a atire1b agb1

triple mutant display defects in root cell elongation specifi-

cally (Figures 5 and 6); such elongation is characterized by

rapid cell-wall synthesis (Verbelen et al., 2006). The biosyn-

thesis and assembly of the plant cell wall relies on the

secretory pathway (Lerouxel et al., 2006). Hence, it is possi-

ble that the AtIRE1A/AtIRE1B- and AGB1-dependent UPR

pathways may be involved in the control of secretory path-

way activities to achieve optimal root cell elongation. To test

this hypothesis, we compared the transcription level of UPR

genes in the root tissue between wild-type, the agb1-3

mutant, the atire1a atire1b double mutant and the atire1a

atire1b agb1 triple mutant using quantitative real-time

RT-PCR. We found that, while only two of ten tested UPR

genes showed lower expression in the root of the atire1a

atire1b double mutant compared to wild-type, transcription

of seven UPR genes was significantly reduced in the atire1a

atire1b agb1 triple mutant compared to wild-type (Figure 7).

These data suggest that the enhanced short-root phenotype

in the atire1a atire1b agb1 triple mutant compared to the

atire1a atire1b double mutant is associated with a lower

abundance of UPR gene transcripts in the root of atire1a

atire1b agb1 triple mutant.

Rel

ativ

e ex

pres

sion

WT atire1a atire1b


0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

** **

**

** *

AtERdj3A AtERdj3B AtERdj2A AtERdj2BBiP1/2 CRT1 CNX1 P58IPK1 PDI6 PDI9

Figure 7. Expression of UPR genes is lower in the root of atire1a atire1b agb1.

Quantitative real-time RT-PCR of BiP1/2, CRT1, CNX1, P58IPK1, PDI6, PDI9, AtERdj3A, AtERdj3B, AtERdj2A and AtERdj2B transcripts in the root tissue of 2-week-old

wild-type, agb1-3, atire1a atire1b and atire1a atire1b agb1. Asterisks indicate significant differences between atire1a atire1b and wild-type or between atire1a atire1b

agb1 and wild-type.

Cell length between400–600 μm

Average lengthof RHI cells

WT atire1a atire1b


atire1a atire1b


WT

* *

Cel

l len

gth

(μm

)

Cel

l len

gth

(μm

)

30

20

10

0

40

(a)

(b) (c)60

20

0

40* *

600 μm

400 μm

(RCJ) 0 μm

WT agb1-3 atire1a atire1b

WT

agb1-3

atire1a atire1b

C atire1a atire1b agb1

1200–1500 (μm)Meristem from RCJ

atire1a atire1b agb1

Figure 6. The elongation zone of atire1a atire1b and atire1a atire1b agb1 roots

is defective.

(a) Confocal microscopy images of roots (longitudinal axis) of wild-type,

atire1a atire1b, atire1a atire1b agb1 and agb1-3 labeled with propidium

iodide. ‘0 lm’ indicates the position of the root cap junction (RCJ).

(b) Mean cell length for wild-type and mutants in the region between 400-600

lm from the RCJ.

(c) Mean cell length for wild-type and mutants in the region showing root hair

initiation (RHI).

Asterisks indicate significant differences between the mutants and wild-type.



DISCUSSION

We have established that AtIRE1A and AtIRE1B are critical for

the plant UPR by showing that loss of function of AtIRE1A

and AtIRE1B leads to over-sensitivity to ER stress and alter-

ation of UPR gene induction. By demonstrating that AtIRE1A/

AtIRE1B and AGB1 independently control the plant UPR, we

have uncovered a genetic interaction between IRE1 proteins

and a G protein-signaling component in the UPR. In addition,

we have shown that AtIRE1A/AtIRE1B and AGB1 contribute

to root growth. Hence, we have identified a new biological

role for the UPR transducers in a multicellular context.

AtIRE1A and AtIRE1B are critical for the plant UPR

IRE1 is essential for growth and development in mammals:

inactivation of the IRE1a gene, encoding one of the two

mammalian IRE1 isoforms, leads to lethality in mouse due to

severe placental dysfunction (Iwawaki et al., 2009). In yeasts,

however, the knockout mutant of the single-copy IRE1 gene

is viable (Cox et al., 1993; Shen et al., 2001). Although the

atire1b-2 mutation is not lethal, it is still not clear whether

AtIRE1B is dispensable for normal growth and development

in plants. A homozygous line of a putative AtIRE1B T-DNA

insertion mutant (SALK_018150, atire1b-1) could not be

isolated after selfing of a heterozygous line (Lu and Chris-

topher, 2008). If the embryonic or reproductive lethality in

atire1b-1 is not caused by an AtIRE1B locus-linked mutation,

then AtIRE1B is an essential gene. This possibility implies

that the atire1b-2 mutant is not a null allele, and this

hypothesis is supported by our data showing that the atir-

e1b-2 mutant is not an RNA null mutant (Figure 1b). In

addition, mammalian IRE1a is an essential gene (Iwawaki

et al., 2009) that mediates diverse biological responses by

physical interaction through its linker and kinase domains

(Hetz and Glimcher, 2009). As the T-DNA insertion site in the

atire1b-2 mutant is located at the end of the kinase domain

(Figure S5), we propose that UPR sensor and kinase function

may be preserved but RNase activity is probably abolished

in the atire1b-2 mutant. Hence, the truncated AtIRE1B pro-

tein in the atire1b-2 mutant may still be able to sense ER

folding homeostasis and transduce the signals through

protein–protein interaction or other unknown mechanisms.

In particular, the partial loss of function of AtIRE1B in the

atire1b-2 mutant may be sufficient to maintain the signaling

responses required for growth and development. Thus, the

atire1b-2 mutation is leaky and the atire1b-2 mutant is via-

ble. In contrast, the T-DNA insertion site in the atire1b-1

mutant is located close to the transmembrane domain

(Figure S4), implying that either membrane insertion of

AtIRE1B or the kinase and RNase activity are affected se-

verely enough to result in complete loss of function of

AtIRE1B in the atire1b-1 mutant. As AtIRE1B may be essen-

tial for embryonic or reproductive development, the null

allele atire1b-1 is lethal.

Although it is still undetermined whether AtIRE1B is an

essential gene in plants, our data clearly show that UPR

activation is compromised in the atire1a atire1b double

mutant (Figure 2), and that both AtIRE1A and AtIRE1B are

critical UPR transducers in plants.

While this paper was under review, Nagashima et al.

(2011) reported a similar ER stress tolerance phenotype of an

ire1a ire1b double mutant using another viable T-DNA

insertion line of AtIRE1B (ire1b-3). The T-DNA insertion in

ire1b-3 is at the N-terminus of the transmembrane domain

(Figure S5), suggesting that the transmembrane and kinase

domains are probably disrupted in ire1b-3. The fact that the

ire1b-3 mutant is viable implies that AtIRE1B may not be an

essential gene. However, whether ire1b-3 is an RNA null

mutant is still uncertain as the transcript analysis of AtIRE1B

in ire1b-3 was only performed using a primer set across the

T-DNA insertion. A complementation test of atire1b-1 with

AtIRE1B will clarify whether AtIRE1B is an essential gene in

plants.

Is bZIP60 the only AtIRE1 substrate in UPR signaling?

It has recently been demonstrated that activation of bZIP60

upon ER stress relies on mRNA splicing by AtIRE1B, but not

by AtIRE1A (Deng et al., 2011). Unlike the atire1a atire1b

double mutant, neither the atire1b-2 nor the bzip60-1 mutant

showed a reduced ER stress tolerance phenotype compared

to wild-type (Figures 2a and 3a). This indicates that AtIRE1A

is sufficient to compensate for the absence of AtIRE1B–

bZIP60 regulation in the plant UPR. Hence, splicing of bZIP60

mRNA may not be the only regulatory mechanism in the

AtIRE1A/AtIRE1B-dependent UPR pathway. Furthermore, as

there were no detectable phenotypic changes in ER stress

tolerance in the bzip60-1 mutant (Figure 3a), we propose that

the ER stress-sensitive phenotype of the atire1a atire1b

double mutant is not simply caused by defects in bZIP60

mRNA splicing. Instead, other target(s) may exist that may be

recognized and activated by either AtIRE1A alone or by both

AtIRE1A and AtIRE1B. In animals and yeasts, only one splic-

ing substrate of IRE1 has been identified and confirmed as a

UPR regulator. However, the existence of multiple splicing

targets of IRE1 in plants may allow them to respond to the

variety of stimuli that they encounter as sessile organisms.

Also, we cannot exclude the possibility that AtIRE1A/AtIRE1B

may regulate the plant UPR through protein–protein inter-

action or other unidentified regulatory mechanisms.

Again, while this paper was under review, Nagashima

et al. (2011) reported that the defect in bZIP60 splicing was

only detected in atire1a atire1b double mutant, but not the

atire1a or atire1b single mutants. Although there is discrep-

ancy in the results regarding bZIP60 mRNA splicing in single

mutants of AtIRE1B (Deng et al., 2011; Nagashima et al.,

2011), the fact that bzip60-1 did not show an ER stress-

sensitive phenotype comparable to that of the atire1a atire1b

double mutant supports the hypothesis that AtIRE1A/



AtIRE1B controls the plant UPR by methods in addition to

splicing of bZIIP60 mRNA.

AGB1 has a positive role in cell survival upon ER stress

Unlike IRE1, a well-characterized UPR sensor in yeasts and

metazoans, the molecular mechanisms of AGB1 regulation

of the UPR are yet unclear. In particular, it is not known that

whether AGB1 controls the UPR through a classic G-protein

signaling function, such as maintaining ion homeostasis

(Hamm, 1998). Sustained ER stress may lead to induction

of the apoptotic pathway. As induction of UPR genes is

higher in the agb1-3 mutant compared to the wild-type

(Figure 4c,d), we propose that AGB1 monitors the induction

level of UPR genes to ensure the UPR is not over-activated,

which may lead to induction of the cell apoptotic pathway.

G-protein components are involved in certain fundamental

cellular functions, including ion homeostasis and cell pro-

liferation, in both plants and animals (Jones and Assmann,

2004). However, to our knowledge, G-protein signaling

pathways have not been reported to regulate the UPR in

metazoans. It is possible that AGB1 has evolved unique

functions in the UPR in plants, but we cannot exclude the

possibility that G-protein signaling pathways are also

involved in the metazoan UPR. Uncovering roles of G-pro-

tein signaling in the metazoan UPR may be challenging due

to the functional redundancy resulting from the presence of

multiple copies of G-protein components in metazoans.

Antagonistic regulation of the plant UPR by AtIRE1A/

AtIRE1B and AGB1

Studies of genetic interactions between UPR regulators in

Caenorhabditis elegans have uncovered complex functional

relationship of UPR regulators (Shen et al., 2005). Estab-

lishing that there are two parallel signaling pathways med-

iated by AtIRE1A/AtIRE1B or AGB1 supports the hypothesis

that a multiplicity of UPR pathways is advantageous for cells

responding to diverse stimuli in multicellular organisms. ER

protein-folding homeostasis is known to be exquisitely

dynamic and to require timely and fine-tuned regulation

(Kaufman, 1999). The evidence that induction of UPR genes

is lower in the atire1a atire1b double mutant but higher in

the agb1-3 single mutant compared to wild-type suggests

that AtIRE1A/AtIRE1B and AGB1 act antagonistically to

maintain a proper UPR (Figure 4c,d). The hypothesis is fur-

ther supported by the fact that the down-regulation of the

AGB1 transcript upon ER stress seen in the wild-type is

disrupted in the atire1a atire1b double mutant.

The effect of AtIRE1A/AtIRE1B and AGB1 on root growth

The UPR is essential for growth and development in mam-

mals. Although we cannot exclude the possibility that AtIR-

E1A and AtIRE1B regulate root growth via a mechanism

unrelated to the UPR, the most likely scenario is that the

AtIRE1A/AtIRE1B- and AGB1-dependent UPR pathways

coordinately contribute to primary root growth. The fact that

the atire1a atire1b agb1 triple mutant displayed enhanced

phenotypes compared to the atire1a atire1b double mutant

in terms of both ER stress sensitivity and root growth defects

supports this hypothesis. Also, the lower transcription level

of UPR genes in the root of the atire1a atire1b agb1 triple

mutant suggests that the shorter root length is due to

compromised secretory capacity (Figure 7). Identification

and characterization of differentially expressed genes in root

tissue between wild-type and the atire1a atire1b agb1 triple

mutant will further help to determine whether the UPR

contributes to root growth, and possibly to define novel

regulatory pathways in root growth and development.

EXPERIMENTAL PROCEDURES

Plant materials and growth conditions

Arabidopsis thaliana ecotype Columbia (Col-0) was used as thewild-type control. The Arabidopsis T-DNA mutants atire1a-4 (WIS-CDSLOX420D09), atire1b-2 (SAIL_238_F07), agb1-3 (SALK_061896),agb1-2 (CS6535), bzip28-1 (SALK_132285), bzip60-1 (SALK_050203)and bip2 (SALK_047956), and the agb1-1 mutant (CS3976) contain-ing a point mutation were obtained from the Arabidopsis BiologicalResource Center (http://abrc.osu.edu/) (Lu and Christopher, 2008;Liu et al., 2007; Wang et al., 2005, 2007). The primers used forgenotyping are listed in Table S1. Surface-sterilized seeds wereplated directly onto square Petri dishes containing half-strengthLinsmaier and Skoog (LS) medium, 1.5% w/v sucrose and 0.4%Phytagel (P8169, Sigma, http://www.sigmaaldrich.com/). For nor-mal growth conditions, plants were grown at 21�C under a 16 hlight/8 h dark cycle.

Tm treatment

In the plate system, tunicamycin (Tm) (T7765, Sigma, http://www.sigmaaldrich.com/; dissolved in DMSO) was directly addedto half-strength LS medium containing 1.5% w/v sucrose and 0.4%Phytagel, at the concentrations indicated. Seeds were directlygerminated in Tm-containing medium for observation of ER stresstolerance. To harvest tissue for UPR gene expression analysis, theseeds were germinated in half-strength LS medium without Tmfor 2 weeks, and then transferred to Tm-containing medium. Inthe hydroponic system (Araponics, http://www.araponics.com/),seedlings were grown in liquid medium (FloraSeries, GHE, http://gb.eurohydro.com/floraseries.html) without Tm for 4 weeks, then10 mg/ml Tm dissolved in DMSO was added to the liquid med-ium. For the infiltration method, a needleless syringe was used toinfiltrate 15 lg/ml Tm into the abaxial side of leaves. As a mockcontrol for the Tm treatment, a volume of DMSO equivalent tothat used to dissolve Tm was used in the same experimentalprocedure.

Genotyping and isolation of multiple T-DNA insertion

mutants

Genotyping of the T-DNA insertion mutants was accomplished bygenomic DNA extraction followed by DNA amplification usingT-DNA- and gene-specific primers. The primers used for genotypingand phenotyping are listed in Table S1. PCR experiments wereperformed under standard conditions using 0.2 mM dNTPs, 0.2 lM

primer and 1 unit of Taq polymerase (Promega, http://www.promega.com/). Homozygous lines for T-DNA insertion of transgenic



plants were isolated by segregation analyses on media containingthe selective marker encoded within the T-DNA. Isolation of multi-allelic lines was performed by performing reciprocal crossesfollowed by genotyping of the F2 generation.

RNA extraction and quantitative RT-PCR analysis

Total RNA was extracted from whole seedlings using an RNeasyplant mini kit (Qiagen, http://www.qiagen.com/) and treated withDNase I (Qiagen). All samples within an experiment were reverse-transcribed at the same time using a high-capacity RNA-to-cDNAmaster mix kit (ABI 4390777, Applied Biosystems, http://www.appliedbiosystems.com/). A ‘no RT’ reaction, in which RNA wassubjected to the same conditions of cDNA synthesis but withoutreverse transcriptase, was included as a negative control in allquantitative RT-PCR assays to ensure the purity of RNA samples.Real-time quantitative real-time RT-PCR with SYBR Green detectionwas performed in triplicate using the Applied Biosystems 7500 fastreal-time PCR system. Data were analyzed by the DDCT method. Thetranscript level was normalized to that of the isopentenyl pyro-phosphate gene (IPP2) for each sample. For AtERdj3A, AtERdj3Band AGB1, the relative transcript level is expressed as the foldchange (mean � SEM) in each genotype under Tm treatment rela-tive to the mock control (set to a value of 1). For BiP3, as the tran-script level was extremely low in the mock control, the relativetranscript level is expressed as the fold change (mean � SEM) inatire1a atire1b at each time point of Tm treatment relative to that inthe wild-type under the same treatment conditions (set to a value of1). For all UPR response genes examined in the root or shoot tissue,the relative transcript level is expressed as the fold change (meanSEM) in each genotype relative to the wild-type under normal

growth conditions (set to a value of 1). We performed three inde-pendent experiments in triplicate. Values presented are means ofthree samples from one representative experiment. A similar pat-tern was observed from three independent biological replicates.

Phenotypical analyses

Root length measurements were averaged from 30 plants for eachgenotype; aerial tissue from ten plants was pooled to estimate thefresh weight. Values were averaged from three individual samplesfor each genotype. Cell length calculations were performed on tenroots for each genotype. Statistical analyses included Student’stwo-tailed t test, assuming equal variance; data with a Pvalue < 0.05 were considered significant.

Arabidopsis stable transformation and complementation

For cloning using the dexamethasone-inducible vector (pTA7002),standard molecular cloning techniques were used. Complementa-tion of agb1-3 was achieved using a 35S–AGB1–YFP fusion. VectorspAtIRE1A-AtIRE1A and 35S-YFP-AGB1 were generated using binaryvectors pGWB1 and pEarlygate104, respectively (Earley et al., 2006;Nakagawa et al., 2007). The genomic or coding regions of geneswere amplified using Gateway-compatible primers from cDNAsynthesized from total RNA of wild-type (Col-0) seedlings usingPhusion high-fidelity DNA polymerase (New England Biolabs,http://www.neb.com/). The PCR fragments were cloned into thedonor vector pDonorTM207 and destination vectors pGWB1 andpEarlygate104. Primer sequences used in this work are listed inTable S1. Arabidopsis plants were transformed by the floral-dipmethod (Clough and Bent, 1998), and transformants were selectedon half-strength LS medium supplemented with hygromycin(20 lg/ml final concentration) and 0.8% w/v agar. Induction ofdexamethasone-inducible clone was achieved using 30 lM dexa-methasone (Sigma).

Confocal laser scanning microscopy

An inverted laser scanning confocal microscope (LSM510 META,Zeiss, http://www.zeiss.com/) was used for imaging analyses.Imaging of propidium iodide-labeled roots (10 lg/ml) was per-formed using 543 nm excitation of a He/Ne laser and an LP 560emission filter. Post-acquisition analyses were performed usingZeiss AIM software. Adobe Illustrator (http://www.adobe.com/) wasused for further image handling.

ACKNOWLEDGEMENTS

We acknowledge support by the Chemical Sciences, Geosciencesand Biosciences Division, Office of Basic Energy Sciences, Office ofScience, US Department of Energy (award number DE-FG02-91ER20021) and the National Aeronautics and Space Agency(NNH08ZTT003N NRA–08-FSB_Prop-0052).

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Genotyping of mutants of AtIRE1A, AtIRE1B and AGB1.Figure S2. No significant differences in BiP3 induction wereobserved in ire1a-4, atire1b-2 or wild-type upon ER stress treatment.Figure S3. Complementation of the Tm sensitivity phenotype ofatire1a atire1b by AtIRE1A or AtIRE1B.Figure S4. No significant differences in cell length were observed inthe root meristems of agb1-3, atire1a atire1b and atire1a atire1bagb1.Figure S5. Diagram of AtIRE1B protein structure.Table S1. Primer list.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

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