Unequally redundant RCD1 and SRO1 mediate stress anddevelopmental responses and interact with transcriptionfactors

Pinja Jaspers1, Tiina Blomster1, Mikael Brosche1, Jarkko Salojarvi1,2, Reetta Ahlfors1, Julia P. Vainonen1, Ramesha A. Reddy1,

Richard Immink3, Gerco Angenent3, Franziska Turck4, Kirk Overmyer1 and Jaakko Kangasjarvi1,*

1Plant Biology, Department of Biological and Environmental Sciences, Viikki Biocenter, University of Helsinki, PO Box 65

(Viikinkaari 1), 00014 Helsinki, Finland,2Department of Information and Computer Science, Helsinki University of Technology, PO Box 5400, FI-02015 TKK, Finland,3Business Unit Bioscience, Plant Research International, 6700 AA Wageningen, The Netherlands,4Max Planck Institute for Plant Breeding Research, Department of Plant Developmental Biology, Carl von Linne Weg 10,

50829 Cologne, Germany

Received 25 February 2009; revised 29 May 2009; accepted 8 June 2009; published online 9 July 2009.*For correspondence (fax 358 9 191 59552; e-mail jaakko.kangasjarvi@helsinki.fi).

SUMMARY

RADICAL-INDUCED CELL DEATH1 (RCD1) is an important regulator of stress and hormonal and developmental

responses in Arabidopsis thaliana. Together with its closest homolog, SIMILAR TO RCD-ONE1 (SRO1), it is the

only Arabidopsis protein containing the WWE domain, which is known to mediate protein–protein interactions

in other organisms. Additionally, these two proteins contain the core catalytic region of poly-ADP-ribose

transferases and a conserved C-terminal domain. Tissue and subcellular localization data indicate that RCD1

and SRO1 have partially overlapping functions in plant development. In contrast mutant data indicate that rcd1

has defects in plant development, whereas sro1 displays normal development. However, the rcd1 sro1 double

mutant has severe growth defects, indicating that RCD1 and SRO1 exemplify an important genetic principle –

unequal genetic redundancy. A large pair-wise interaction test against the REGIA transcription factor

collection revealed that RCD1 interacts with a large number of transcription factors belonging to several

protein families, such as AP2/ERF, NAC and basic helix–loop–helix (bHLH), and that SRO1 interacts with a

smaller subset of these. Full genome array analysis indicated that in many cases targets of these transcription

factors have altered expression in the rcd1 but not the sro1 mutant. Taken together RCD1 and SRO1 are

required for proper plant development.

Keywords: ROS, unequal genetic redundancy, protein–protein interaction, transcriptional regulation, WWE

domain, DREB2A.

INTRODUCTION

The RCD1 gene (At1g32230; also known as CEO1) was first

isolated for its ability to complement sensitivity to reactive

oxygen species (ROS) in yeast (Belles-Boix et al., 2000).

Forward genetic screens for ozone sensitivity and paraquat

tolerance led to the isolation of the Arabidopsis rcd1-1 and

rcd1-2 mutants (Overmyer et al., 2000; Fujibe et al., 2004).

RADICAL-INDUCED CELL DEATH1 (RCD1) protein defines a

six-member family of proteins in Arabidopsis, which have

been named SIMILAR TO RCD-ONE (SRO). The paralogous

SRO1 (At2g35510) is the closest homolog of RCD1; they are

61% identical and 74% similar, sharing the same conserved

features (Figure 1). They both have canonical nuclear

localization sequences and two highly conserved protein

domains: the WWE domain (PS50918; Aravind, 2001) and a

poly(ADP-ribose) polymerase (PARP)-like ADP-ribose trans-

ferase catalytic domain (PS51059). SRO2–SRO5 are shorter

proteins, lacking the N-terminal WWE domain and the

nuclear localization signals.

The WWE domain is a globular domain predicted to

mediate protein–protein interactions (Aravind, 2001). This

has been demonstrated for the WWE domains of the

Drosophila deltex protein; these interact with ankyrin

repeats of the Notch receptor (Zweifel et al., 2005). The

majority of WWE proteins are related to the protein ubiquitin

268 ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd

The Plant Journal (2009) 60, 268–279 doi: 10.1111/j.1365-313X.2009.03951.x

transfer complex E3 ligase and contain conserved E3

functional domains such as the RING, RING-H2 and HECT.

RCD1 and SRO1 represent a second, PARP-domain-bearing,

class of WWE proteins (Aravind, 2001). It was proposed that

the ubiquitin ligase- and PARP-related WWE proteins could

bind the same targets and decorate them with alternative

modifications, resulting in different functional conse-

quences (Aravind, 2001). However, no experimental evi-

dence supporting this has so far been found. Arabidopsis

RCD1 and SRO1 and their orthologs in other species are

plant-specific proteins, and are the only known WWE

domain proteins in plants. However, proteins with similar

WWE and PARP domain architecture are found in other

eukaryotes, even as distant as humans (Otto et al., 2005;

Hassa et al., 2006).

The PARP domain in RCD1 and SRO1 is classified as a

partial PARP catalytic domain of the ADP-ribosylating

superfamily (cl00283) containing the conserved NAD-bind-

ing signature (Ame et al., 2004). The PARP domain is found

in association with several other domains, for example

zinc fingers, Macro, ankyrin or the WWE (Ame et al., 2004).

Typically the targets of ADP-ribosylation reactions are

proteins, and ADP ribosylation/deribosylation may affect

their activity or stability, but low molecular weight mole-

cules can also be targeted. Poly(ADP-ribose) polymerases

are involved in a wide variety of cellular functions, including

response to DNA damage, programmed cell death, regula-

tion of gene expression and telomere length (Ame et al.,

2004; Diefenbach and Burkle, 2005).

The RCD1–SRO gene family members have a role in stress

responses. The sro5 mutant is sensitive to salt and oxidative

stress (Borsani et al., 2005). RCD1 is involved in salt stress by

physical interaction with SOS1, a plasma membrane Na+/H+

antiporter (Katiyar-Agarwal et al., 2006). Plants lacking RCD1

function have many and varied phenotypes in addition to

changes in sensitivity to ROS. These include altered nitric

oxide, jasmonic acid (JA) and ethylene responses, as well as

developmental phenotypes such as early flowering and

alterations to leaf and rosette morphology (Overmyer et al.,

2000; Ahlfors et al., 2004, 2009). These phenotypes suggest

that RCD1 can serve as an integration point between

hormone signaling and a coordinated ROS response.

Mechanistically, little is known about RCD1, and espe-

cially RCD1-related proteins but their domain architecture

suggests a conserved function. Therefore, we have concen-

trated on the two WWE-containing members of the protein

family, RCD1 and SRO1, to assess their importance and

possible functional divergence. Here we show that RCD1

interacts with several transcription factors (TFs) which are

involved in both developmental and stress-related pro-

cesses reflecting the phenotypes of rcd1 mutants. Further-

more we show that both RCD1 and SRO1 are required for the

proper development of Arabidopsis in an unequally redun-

dant manner.

RESULTS

Interaction partners of RCD1 and SRO1 proteins

In order to probe the function of the RCD1 and SRO1

proteins, yeast two-hybrid screening was undertaken using

full-length RCD1 and SRO1 proteins as baits. Eleven unique

RCD1 and four SRO1 interaction partners were isolated

(Table 1). The majority of them (7 out of 11, Table 1) were

TFs belonging to the DREB, NAC, basic helix–loop–helix and

constans-like zinc finger-containing families. The predomi-

nant interaction partner that was isolated was DREB2A,

a TF that belongs to the AP2/ERF TF family and has a central

role in the ABA-independent pathway in acclimation to salt

and osmotic stress and high temperatures (Sakuma et al.,

2006; Yamaguchi-Shinozaki and Shinozaki, 2006). DREB2A

represented about 1000 of the 1200 colonies isolated with

RCD1 and it was also the predominant clone among the 370

colonies discovered with SRO1.

To overcome the technical difficulties presented by the

abundance of DREB2A and to address the specificity of

interaction within the TF families, we utilized the REGIA

collection of 1394 Arabidopsis TFs (http://www.jicgenom

elab.co.uk/libraries/arabidopsis/tf.html; (Paz-Ares and The

REGIA Consortium, 2002; FT, manuscript in preparation).

This allowed high-throughput pair-wise interaction tests

against RCD1 and SRO1. Screening of the REGIA collection

confirmed that RCD1 interacts with a large number of TFs

(Table 1). Of the seven TFs isolated in the library screen, all

five present in the REGIA collection were positive for

interaction with RCD1 (Table 1). In order to be included in

RCD1

SRO1

N C

0 100 200 300 400 500 aa

55% 70% 59%

WWE P ARP RST

rcd1–4

sro1-1

rcd1–3rcd1–2rcd1–1

NLS

Figure 1. Structure of the RADICAL-INDUCED CELL DEATH1 (RCD1) and

SIMILAR TO RCD-ONE1 (SRO1) proteins.

The protein structure of RCD1 and SRO1 showing the location of the predicted

functional features, mutations and T-DNA insertions. Percentages indicate the

proportion of identical amino acids between the corresponding domains of

the two proteins. Abbreviations: NLS, nuclear localization signal; WWE, WWE

domain (PS50918); PARP, poly(ADP-ribose) polymerase catalytic region

(PS51059); RST, RCD1-SRO-TAF4 domain (amino acids 494–569 for RCD1

and 493–568 for SRO1).

RCD1 and SRO1 redundancy 269

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Table 1, the interaction partners were required to show

growth in the yeast two-hybrid screen with full-length RCD1

or SRO1 on both –Ade and –His selection, and to be positive

for b-galactosidase assay. Of the total of 46 RCD1-interacting

and 3 SRO1-interacting TFs identified, the 19 listed in Table 1

met these stringency criteria. Several other proteins not

meeting these strict criteria were also isolated, including

proteins involved in processes known to be relevant to rcd1

mutant phenotypes, such as PIF3, MYC2 and COL9 (very

similar to strongly RCD1-interacting COL10) which only

interacted with truncated RCD1 lacking the WWE domain. A

subset of these observed interactions was verified in an

in vitro system using recombinant RCD1 and glutathione-

S-transferase (GST)-fused TFs. Recombinant RCD1 and

four representative RCD1-interacting proteins, each from a

distinct TF family, were bacterially expressed, purified and

tested. Capture of DREB2A–GST, STO–GST and COL10–GST

with GSH beads and subsequent detection in a western blot

with a peptide anti-RCD1 antibody directed against the RCD1

C-terminus resulted in the expected RCD1 band of 65.7 kDa

(Figure S1 in Supporting Information). Additionally, DRE-

B2A–GST exhibits a smaller approximately 47 kDa band

which probably represents an RCD1 degradation product.

Exceptionally, the MYB91–GST band appeared as a high

molecular weight aggregate. Lack of signal in the GST

control (Figure S1) and the unpurified bacterial lysate (not

shown) indicates the specificity of the assay. We conclude

that the interaction of RCD1 with DREB2A–GST, STO–GST,

MYB91–GST and COL10–GST was confirmed in this in vitro

binding assay.

To map interaction domains, RCD1 truncations were

created and assayed with partners identified in the REGIA

screen. Interaction was assayed with full-length RCD1 pro-

tein (FL), a C-terminal truncation, WWE plus PARP (WP)

equivalent to the predicted truncated proteins in rcd1 mutant

alleles (Figure 1) and an N-terminal truncation PARP plus C-

terminus (PCT) resembling the shorter gene family mem-

bers SRO2–SRO5 (Table 1). In all cases the RCD1 C-terminus

Table 1 RADICAL-INDUCED CELL DEATH1 (RCD1) and SIMILAR TO RCD-ONE1 (SRO1)-interacting proteins. List of proteins interacting withRCD1 and/or SRO1 in the yeast two-hybrid system. A stress-enriched cDNA library was screened with both proteins as baits and a large-scalepair-wise screen was performed against the REGIA transcription factor collection

Name AGI TF

RCD1 SRO1

LIB

REGIA

LIB REGIAFL WP PCT

DREB2A At5g05410 Y + + ) + + +DREB2B At3g11020 Y + + ) + + +DREB2C At2g40340 Y ) + ) + ) +ANAC013 At1g32870 Y + + ) + ) )ANAC046 At3g04060 Y ) + ) + ) )ANAC082 At5g09330 Y + NA NA NA + NASTO At1g06040 Y ) + ) + ) )COL10 At5g48250 Y + + ) + ) )PIF5 At3g59060 Y ) + ) + ) )PIF7 At5g61270 Y + + ) + ) )UNE10, similar to PIF7 At4g00050 Y ) + ) + ) )AtbHLH011 At4g36060 Y ) + ) + ) )AtbHLH019 At2g22760 Y ) + ) + ) )ILR3, bHLH At5g54680 Y ) + ) + ) )IAA11 At4g28640 Y ) + ) + ) )MYB91, AS1 At2g37630 Y ) + ) + ) )bZIP At2g16770 Y ) + ) + ) )TGA2, AHBP-1B At5g06950 Y ) + ) + ) )WRKY47 At4g01720 Y ) + ) + ) )GT2-related At2g33550 Y ) + ? ? ) )AtIDD5 At2g02070 Y + NA NA NA ) NAATCSLA9, RAT4 At5g03760 N + NA NA NA ) NAViniculin/a-catein domain containing At4g09060 N + NA NA NA ) NAExpressed protein At5g08720 N + NA NA NA + NAExpressed protein At1g19835 N + NA NA NA ) NA

Abbreviations: TF, transcription factor; LIB, result of the library screen (LexA-based); REGIA, results of the pairwise interaction test (GAL4-based);FL, full-length RCD1; WP, truncated RCD1 protein containing only the WWE and poly(ADP-ribose) polymerase (PARP) domains (amino acids1–471); PCT, truncated RCD1 protein containing only the PARP and C-terminal domain (amino acids 241–589); NA, not available in the REGIAcollection.

270 Pinja Jaspers et al.

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 268–279

was required for interactions (Table 1). SRO1 exhibited the

same pattern with its three interaction partners from the

REGIA interaction test.

RCD1 and SRO1 localization

Tissue-level localization of RCD1 and SRO1 was determined

by GUS staining visualizing the distribution of promoter

activities with promoter::UidA fusions (Figure 2). RCD1 and

SRO1 promoters exhibited similar activity patterns. Their

expression was highest in young developing tissues, such

as young leaves and root tips (Figure 2a,b,k,l). In fully

developed mature tissue RCD1 and SRO1 promoters were

active in vascular and perivascular tissues in the veins of

leaves, petioles and the root vascular column (Figure 2c–

f,i,j). Additionally, in leaves, RCD1, and to a lesser extent

SRO1, promoter activity was visible in stomatal guard cells

(Figure 2g,h).

Subcellular localizations of RCD1 and SRO1 were inves-

tigated by transient expression of yellow fluorescent protein

(YFP) fusions in onion epidermal cells. In Figure S2 both

RCD1 and SRO1 were located exclusively in the nucleus,

while the YFP control was located in both nucleus and

cytoplasm.

Mutant analysis

In addition to the two ethane methyl sulfonate (EMS) alleles,

rcd1-1 and rcd1-2, which both result in a premature stop

codon and thus predicted truncated proteins (Overmyer

et al., 2000; Fujibe et al., 2004), we characterized the RCD1

insertion alleles rcd1-3 and rcd1-4, and the only known SRO1

allele sro1-1, which has a T-DNA-insertion close to the 3¢ end

of the coding sequence. The position of these mutations

relative to RCD1 and SRO1 protein features is depicted in

Figure 1. The transcripts present in these mutants were

assayed by real-time quantitative (q)PCR (Figure S3). The

rcd1-3 and sro1 mutants result in predicted truncated

proteins; only rcd1-4 is a transcriptional null.

The mutant phenotypes of rcd1 have been previously

characterized (Overmyer et al., 2000; Ahlfors et al., 2004;

Fujibe et al., 2004; Katiyar-Agarwal et al., 2006) and include

alterations in stress sensitivity, gene expression and devel-

opment (Table S1). Generally, all rcd1 alleles are identical in

phenotype. The sro1 mutant, on the other hand, is visually

indistinguishable from wild type and has, if any, only a few

subtle phenotypes, which are opposite to those of rcd1

(Table S1, Figure 3; Sachin Teotia and Rebecca S. Lamb,

Ohio State University, USA, personal communication).

To investigate the functional redundancy of RCD1 and

SRO1, an rcd1 sro1 double mutant was created. Initial

screens of rcd1 · sro1 F2 populations grown on soil failed to

identify double mutants; plants with a growth phenotype

more severe than rcd1 were present but genotyping

revealed they were rcd1)/)sro1+/). Germination and growth

in sterile culture on sugar-containing media enabled the

rescue of the rcd1 sro1 double mutant, which was confirmed

by PCR genotyping (not shown). The double mutant had

an extremely stunted phenotype and rcd1)/)sro1+/) had an

PRCD1:UidA PSRO1:UidA

(a) (b)

(c) (d)

(e) (f)

(g) (h)

(i) (j)

(k) (l)

Figure 2. RCD1 and SRO1 promoter activity.

The expression of b-glucuronidase fused with the promoter fragment of RCD1

and SRO1 genes (left and right column, respectively) was examined in

different plant tissues of 14-day-old plants: (a), (b) rosette; (c), (d) leaf

mesophyll tissue; (e), (f) vascular tissue; (g), (h) guard cells; (i), (j) root; (k), (l)

root tip. Scale bar = 500 lm.

RCD1 and SRO1 redundancy 271

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 268–279

intermediate stunted phenotype (Figure 3a). These plants,

when rescued by germination on plates, could be trans-

planted to soil and grown to maturity (Figure 3b). However,

the double mutant remained stunted, had deformed leaves

and other developmental defects, and typically produced

only one or two siliques resulting in very few seeds. This

indicates a redundant function for RCD1 and SRO1 that is

essential for plant development.

Gene expression analysis

Microarray hybridization was performed with rcd1 and sro1

mutant alleles. All alleles of rcd1 had similar gene expres-

sion profiles, thus data analysis was performed with data

pooled from all of them (see Table S2 and Appendix S1). In

total, 517 genes exhibited altered expression in rcd1 (252

with increased expression, 265 with reduced expression),

whereas sro1 had only one gene (At5g11330) with a signif-

icant difference in regulation compared with wild type (Col-

0). However, differential expression of this gene in sro1

could not be verified by real time quantitative PCR (qPCR;

data not shown). In addition, expression of several other

genes, with significance close to the P < 0.05 cut-off, was

tested with qPCR and only one of these, a thylakoidal APX

(tAPX; At1g77490), exhibited consistently lower mRNA

abundance in sro1 in comparison to the wild type (Table 2).

Fifteen marker genes from several functional categories

were selected for microarray data validation by qPCR

(Table 2). Marker genes related to salicylic acid (SA)

signaling, DREB2A target genes and stress, all processes

specifically related to rcd1 phenotypes, were used for

confirmation. In general the array and qPCR data were in

agreement, but with slightly larger fold changes for some

genes in the qPCR assay (Table 2).

The literature and gene expression databases were mined

for data identifying the target genes of RCD1-interacting TFs

(Table 1), or processes known to be affected in rcd1. When

available, these data were used to test for gene enrichment

within the differentially expressed genes in rcd1. The control

(i) (ii)

(iii) (iv) (v)

(i) (ii) (iii) (iv)

(v)

(a)

(b)

Figure 3. Mutant growth phenotypes.

(a) Three-week-old plants grown in sterile culture.

(b) Four-week old plants grown in soil under normal growth conditions: (i) Col

WT (ii) rcd1-4, (iii) sro1, (iv) rcd1 sro1, (v) rcd1)/) sro1)/+. Scale bar = 1 cm.

Table 2 Real-time quantitative-PCR analysis with qPCR selected marker genes in rcd1 alleles and sro1. qPCR on selected marker genes in 3-weekold Col-0, rcd1-1, rcd1-2, rcd1-3, rcd1-4 and sro1. The fold ratios for the mutants is calculated to the wild-type Col-0. For a comparison the resultof the array analysis is also presented. The DREB2A targets were extracted from Sakuma et al. (2006). All experiments were repeated at leastthree times, one representative experiment is shown. Values for tAPX, the only gene consistently altered in sro1, are highlighted in bold

AGI code Annotation Col-0

rcd1-1 rcd1-2 rcd1-3 rcd1-4 sro1DREB2AtargetPCR Array PCR PCR Array PCR Array PCR Array

Cold/dehydration regulatedAt2g42540 COR15A 1.0 0.2 0.6 0.3 0.4 0.3 0.3 0.5 1.7 1.5 YesAt5g52310 LTI78 1.0 0.6 0.7 0.5 0.5 0.6 0.5 0.6 1.0 0.9 YesAt5g15970 KIN2/COR6.6 1.0 0.3 NA 0.4 0.3 NA 0.3 NA 0.9 NA YesAt3g56080 Dehydration-responsive 1.0 0.6 0.6 0.5 0.5 0.8 0.5 0.8 0.8 1.2

Transcription factorsAt2g30250 WRKY25 1.0 5.2 3.7 5.7 5.4 4.1 5.8 4.0 0.9 1.6At2g40750 WRKY54 1.0 0.4 0.8 0.3 0.2 0.6 0.3 0.7 0.7 1.1At3g56400 WRKY70 1.0 0.4 0.9 0.5 0.3 0.9 0.4 0.9 0.7 1.4 YesAt1g49010 MYB 1.0 0.3 NA 0.2 0.4 NA 0.2 NA 0.8 NAAt5g18270 ANAC087 1.0 14.8 2.5 14.7 17.3 5.8 18.6 3.2 0.8 NA

Stress or defense relatedAt3g22370 AOX1a 1.0 24.8 12.7 28.5 28.8 19.4 27.7 17.3 1.1 1.0At5g43450 ACC oxidase 1.0 29.8 2.5 36.5 32.4 8.5 37.4 10.0 1.1 NAAt2g14610 PR-1 1.0 0.02 NA 0.1 0.1 0.8 0.02 NA NR NAAt1g77490 tAPX 1.0 0.7 0.9 0.7 0.7 1.0 0.7 0.8 0.4 0.6At2g21640 UPOX 1.0 58.9 21.0 68.0 64.1 32.8 64.7 26.4 1.2 1.5At4g12500 EARL1 family 1.0 0.7 0.7 0.7 0.8 0.6 0.5 0.8 0.8 1.5 Yes

NR, no reproducible data could be obtained for this gene; NA, not available.

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comparison of target genes for two TFs not interacting with

RCD1, HY5 and LEC2 (Braybrook et al., 2006; Lee et al.,

2007), did not show significant enrichment (P = 0.26 and

0.19, respectively) in the rcd1 differentially regulated genes

(Table S2). DREB2A target genes identified in Sakuma et al.

(2006) were significantly over-represented in the list of

genes with altered regulation in the rcd1 mutant (Table S2;

P = 3.76 · 10)6). Furthermore, the qPCR data validated lower

expression levels of several DREB2A target genes in rcd1

(Table 2).

Several RCD1-interacting TFs (PIF5, PIF7, COL9, COL10

and STO) belong to families known to play a role in light

responses (Castillon et al., 2007; Indorf et al., 2007).

Experimental data identifying target genes of these TFs

are not available. However, for PIF3 (a close family

member to PIF5 and PIF7), which interacted with the

truncated RCD1 lacking the WWE domain, there is a data

set describing PIF3-dependent red light-regulated genes

(Monte et al., 2004). These genes were significantly

enriched in the rcd1 gene expression data (Table S2;

P = 1.25 · 10)3). This suggests that RCD1 could be

involved in light responses.

The RCD1-regulated genes exhibited a statistically sig-

nificant enrichment of genes that are expressed to lower

levels in 35S:MYC2 (Table S2; P = 5.23 · 10)6). MYC2 was

positive for interaction with RCD1 in the REGIA screen,

however, only with the truncated RCD1 lacking the WWE

domain (PCT). MYC2 is a regulator of ABA and JA

responses (Lorenzo et al., 2004) and is required for para-

quat tolerance (Dombrecht et al., 2007), which relate to rcd1

phenotypes.

RCD1 interacted with TGA2 (Table 1), a regulator of SA-

responsive genes (Zhang et al., 2003). We tested the expres-

sion of several known TF regulators of SA responses as well

as the classical SA marker gene PR1 with qPCR (Table 2).

WRKY25, a negative regulator of the SA response (Zheng

et al., 2007), had increased expression in rcd1. In contrast

the SA-positive regulators WRKY54 and WRKY70 (Li et al.,

2004) and the downstream response gene PR1 had lower

expression levels.

The expression data set was further analyzed for enrich-

ment of gene ontology (GO) classes in rcd1 (Table S3).

Among the most enriched biological processes were abiotic

stress, cold, temperature, osmotic stress, salt and water

deprivation. The cellular component classifications most

enriched were the extracellular region, proteasome com-

plex, apoplast and cell wall.

Promoter swap and complementation

To further dissect the individual functions of RCD1 and

SRO1, promoter swap and complementation studies were

undertaken for three representative rcd1 phenotypes. We

tested the ability of RCD1 driven by its own promoter and

SRO1 driven by the RCD1 promoter to complement two

alleles of rcd1 and assessed flowering time, the expression

of several marker genes and paraquat tolerance (Figure 4,

Table S4).

In all experiments, under the control of its own promoter,

RCD1 fully complemented both rcd1-2 and rcd1-4 for all

three phenotypes tested (Figure 4a–c, Table S4). In contrast,

there was great variation in the ability of SRO1 to function-

ally replace RCD1. The rcd1 early flowering phenotype was

fully or nearly fully complemented by SRO1 (Figure 4a).

Varied degrees of partial complementation were seen for the

expression of the marker gene ANAC087 (Figure 4b), as well

as other marker genes (Table S4). However, SRO1 provided

no functional compensation for rcd1 in the paraquat toler-

ance assay (Figure 4c).

As a control, RCD1 and SRO1 protein levels were analyzed

from wild-type, rcd1 and sro1 mutants, complemented rcd1

mutant, and promoter swap lines by western blot using an

RCD1-specific antibody (Figure 4d). Surprisingly, neither the

wild type nor the transgenically expressed proteins could be

detected, even when full complementation of mutant

phenotypes was achieved (Figure 4a–c). Detection was

tested with two different peptide antibodies targeted for

the N- and C-termini of RCD1, antibodies raised against

denatured and native full-length recombinant RCD1, as well

as a commercial antibody against the hemagglutinin

(HA)-tag present in the complementation constructs (data

not shown). Additionally, anti-GFP and anti-Myc antibodies

were tested to detect a constitutively expressed tagged

RCD1 in several independent plant lines, with the same

result (not shown). A bacterially produced recombinant

RCD1 protein dilution series (Figure 4e) demonstrated that

5 ng of RCD1 protein could be detected with the antibody

used in Figure 4(d).

RCD1 and SRO1 conserved C-terminus

The results presented above suggest the importance of the

C-terminal regions of RCD1 or SRO1. Alignment of RCD1 and

SRO1 proteins with RCD1-like sequences from grape, pop-

lar, rice and Physcomitrella identified a highly conserved

domain at the C-terminal end of these proteins corre-

sponding to amino acids 494–569 in RCD1 and 493–568 in

SRO1 (Figure 5). It also bears homology to TBP-ASSOCI-

ATED FACTOR 4 (TAF4) and TAF4b, which are components

of the transcription initiation factor complex TFIID (Lago

et al., 2004). This region of TAF4 that is related to RCD1 and

SRO1 is, however, different from the conserved TAF4

superfamily-defining domain (Pfam 05236). This new

domain is named the RST (RCD1 SRO TAF4) domain and has

been registered with Pfam.

DISCUSSION

Unequal genetic redundancy between two homologous

genes refers to a situation where the loss of function of one

gene has a strong phenotype and the loss of function of the

RCD1 and SRO1 redundancy 273

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second gene has no distinguishable phenotype, but in the

double mutant phenotypes are strongly enhanced and even

novel phenotypes may appear (Briggs et al., 2006). Exam-

ples of unequal genetic redundancy in Arabidopsis include

the two TFs HY5 and HYH regulating photomorphogenesis

(Briggs et al., 2006) and two isochorismate synthases ICS1

and ICS2, where ICS1 has the major activity and lack of ICS2

is only noticeable in an ics1 background (Garcion et al.,

2008). RCD1 and SRO1 present a new example of this

genetic principle. Similar promoter activity and subcellular

localization of RCD1 and SRO1 indicate that they might share

redundant functions (Figures 2 and S3). Direct proof for the

unequal redundancy of RCD1 and SRO1 comes from the

analysis of the corresponding single, double and heterozy-

gous double mutants (Figure 3); rcd1 has strong develop-

mental phenotypes whereas sro1 resembles the wild type. In

crosses between these mutants the developmental pheno-

types were enhanced, resulting in severe growth defects in

the rcd1 sro1 double mutant. After gene duplication it is

assumed that one of the redundant copies is free of selective

restraint and could assume a new function. The promoter

swap data (Figure 4) demonstrated that SRO1 was able to

complement RCD1 function in inhibiting flowering, partially

restoring changes in gene expression, but had no effect on

paraquat responses when its expression was driven by the

RCD1 promoter. This indicates that SRO1 still has some – but

not all – of the RCD1 functions.

This shared function of RCD1 and SRO1 could be related

to their ability to interact with TFs. All SRO1 interaction

partners also interacted with RCD1, indicating at least partial

redundancy of the two proteins (Table 1). The RCD1 func-

tions not shared with SRO1 include those leading to

developmental changes, ozone sensitivity and paraquat

tolerance, most of the TF interactions and changes in gene

expression in the rcd1 mutant. One interpretation would be

that the unique functions of RCD1 come from its ability to

interact with more and different TFs. In other words, RCD1

(c)

Col 0

rcd1–2 rcd1–4 —

Control1 M PQ

Chl

Ac

onte

nt (

mg/

leaf

dis

k)

0.0

0.2

0.6

0.4

(b)

Col 0

rcd1–2 rcd1–4—

—

ANAC087

5

10

15

20

Rel

ativ

e ex

pres

sion

(d)

rcd1–2 rcd1–4

Col 0 —

—

25

30

20 0

Bud

e m

erge

nce

(day

s)(a)

Col 0

sro1

rcd1–2 rcd1–4

P RDC1:R

CD1

P RCD1:S

RO1 —

—

P RCD1:R

CD1

P RCD1:S

RO1

—

(e) 50 20 10 5 2 1

RCD1

ng RCD1 6 × His

95% CI

RCD1

Amido black

50 n

g RCD1

6 ×

His

a

b

a a,c a

b

a a

a,c

a

b b

c

d

c

d

95% W ald CI

a a

a a

b

b b

b

sro1

P RDC1:R

CD1

P RCD1:S

RO1

P RCD1:R

CD1

P RCD1:S

RO1

sro1

P RDC1:R

CD1

P RCD1:S

RO1

P RCD1:R

CD1

P RCD1:S

RO1

sro1

P RDC1:R

CD1

P RCD1:S

RO1

P RCD1:R

CD1

P RCD1:S

RO1

Figure 4. Complementation and promoter swaps.

(a) Flowering times in rcd1 and sro1 mutants and complementation lines.

Genotypes that do not differ significantly from each other are marked with the

same letter. Significance was determined using one-way ANOVA with Tukey

honestly significant differences (HSD) post-hoc analysis. Error bars indicate

standard deviation and the scale bar 95% confidence interval.

(b) Transcript levels of an example gene that has altered expression in rcd1

mutants by quantitative real-time PCR. Genotypes denoted by the same letter

do not differ significantly from each other, according to one-way ANOVA with

Tukey HSD post-hoc analysis. Transcript levels of additional genes are

available in Table S4.

(c) The paraquat sensitivity of rcd1 and sro1 mutants and complementation

constructs. Error bars indicate standard deviation and the scale bar the 95%

Wald confidence interval. Genotypes marked with the same letter respond

similarly to the paraquat treatment. Analysis was carried out with generalized

linear model using the Wald statistic for hypothesis testing and applying

Bonferroni correction to the P-values.

(d) Western detection of RADICAL-INDUCED CELL DEATH1 (RCD1) protein

with a specific antibody. The black bar indicates the 70-kDa marker band.

(e) Detection limit of RCD1 by the anti-RCD1 antibody used in panel (d).

274 Pinja Jaspers et al.

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 268–279

and SRO1 could have the same biochemical function and

differ only in their efficiency and/or protein interactions to

perform this function.

Yeast two-hybrid library screens identified a number of

RCD1- and SRO1-interacting proteins, the majority of which

are TFs, confirming previous studies that identified DREB2A

and STO as RCD1-interacting proteins (Belles-Boix et al.,

2000). Interaction partners of RCD1 represented different TF

families. In several cases RCD1 interacted with multiple

family members, for example DREB2A, DREB2B and

DREB2C or ANAC013, ANAC046 and ANAC082. However,

when considering the entire REGIA collection (FT,

manuscript in preparation) tested here, there was specificity

because RCD1 only interacted with certain TF family mem-

bers, as in the examples above – 3 out of 56 DREBs and 3 of

66 ANACs tested (Table 1).

Based on the western analyses with several antibodies,

RCD1 apparently acts in plant tissues in very minute

quantities or in specific cells, and thus has an extremely

low concentration in protein extracts. This makes the

in planta verification of these interactions difficult and is

further complicated by the fact that most RCD1 interaction

partners are TFs that are likely to be under strict regulation.

This is exemplified by PIF3 and PIF5, which are detectable

only for a short time after the beginning of the light period

(Al-Sady et al., 2006; Shen et al., 2007), and by DREB2A in

which a negative regulatory domain keeps the protein levels

low under most conditions (Sakuma et al., 2006). This

suggests that RCD1 and its interaction partners may be

regulated at the level of protein stability. To overcome this

we used in vitro binding experiments with bacterially

produced proteins to validate yeast two-hybrid interaction

results. In these experiments, we chose interaction partners

from four representative TF families. These results

(Figure S1) demonstrate that RCD1 binds DREB2A, STO,

MYB91 and COL10 in vitro. As a pre-condition to in planta

verification of these interactions, further studies will be

required to identify tissues, conditions or the genetic

background in which RCD1 is stabilized. The presence of

RCD1 and its interactions at only low, tightly regulated levels

at specific times in plant development suggests that it may

be involved in a pre-conditioning of plant signaling config-

uration that has lasting implications throughout the plant’s

life. This is consistent with the observation that RCD1 is

expressed primarily in juvenile leaf tissue (Figure S3) yet

has dramatic phenotypes in adult leaf tissues (Figure 3,

Table S1).

Additionally, several lines of evidence support the

biological significance of the interaction of RCD1 with TFs.

In the cases for which the function of RCD1 interaction

partners is known, these functions are consistent with the

processes altered in rcd1 mutants. For example, DREB2A,

DREB2B, DREB2C and the previously isolated RCD1 interac-

tion partner SOS1 (encoding a Na+/H+ antiporter) are

involved in responses to salt and osmotic stress (Figure 6)

and the rcd1 mutant is salt sensitive (Katiyar-Agarwal et al.,

2006). DREB2A and ANAC013 expression is regulated by UV-

B radiation (Safrany et al., 2008), consistent with the UV-B-

resistant phenotype of rcd1 (Fujibe et al., 2004). ILR3, IAA11

and MYB91/AS1 participate in auxin signaling (Figure 6),

which could be involved in the developmental changes seen

in rcd1 such as decreased apical dominance, altered leaf

shape and changes in rosette architecture (Table S1), which

resemble the phenotype of the as1 mutant (Byrne et al.,

2000). Interestingly, RCD1 also interacted with STO, COL10,

PIF5 and PIF7 (Figure 6), proteins related to light signaling

(Castillon et al., 2007; Indorf et al., 2007). The large number

of light-related interaction partners suggests a function for

RCD1 in light responses, and preliminary phenotypic

analysis of rcd1-4 under different light conditions supports

this suggestion (G. Neuhaus and M. Rodrıguez-Franco,

University of Freiburg, Germany, personal communication).

Analysis of known target genes of the TFs interacting with

RCD1 provides further evidence supporting the biological

relevance of the RCD1 interactions seen in this study. In the

case of the RCD1-interacting proteins DREB2A, MYC2 and

Figure 5. RADICAL-INDUCED CELL DEATH1 (RCD1) and SIMILAR TO RCD-ONE1 (SRO1) C-terminal alignments.

Alignments defining the conserved RST (RCD1 SRO TAF4) domain. Sequences aligned were Arabidopsis RCD1, SRO1, TAF4 and TAF4b. Remaining sequences were

RCD1 and TAF4 from the indicated species. Abbreviations and sequences used: AthRCD1, Arabidopsis thaliana gene At1g32230; AthSRO1, A. thaliana gene

At2g35510; PtRCD1, Populus trichocarpa, eugene3.00030694; VvRCD1, Vitis vinifera, gi|147815590; PpRCD1, Physcomitrella patens, gi|162690773; OsjRCD1, Oryza

sativa japonica, gi|125533074; Ath TAF4, A. thaliana gene At5g43130; AthTAF4b, A. thaliana gene At1g27720; VvTAF4, V. vinifera, emb|CAN80359.1|; PtTAF4,

P. trichocarpa, gw1.XIV.649.1; OsjTAF4, O. sativa japonica, Os02g0654400; PpTAF4, P. patens, gb|EDQ59859.1|. Grey boxes indicate similarity and black boxes

indicate identity. The Physcomitrella sequences contain amino acid insertions not present in other species; in PpRCD1 between E551 and M590

(RLLHAIKLLQGSQINMKLYITWPAFCSAIVLTLHQFSRF) and in PpTAF4 between K271 and Q277 (ASFGA). These were removed from the alignment.

RCD1 and SRO1 redundancy 275

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 268–279

PIF3, the ‘footprint’ of genes known to be their downstream

targets is visible among the genes with altered expression in

the rcd1 mutant (Table S2). These target genes had both

higher and lower expression in rcd1, thus a simple model in

which RCD1 would be only a positive or a negative regulator

of TF activity is too simplistic. A more likely scenario is that

RCD1 function is context dependent, so under certain

conditions it has a positive effect and in others a negative

one. One example of where RCD1 could be a positive

regulator of TF function is seen in the DREB2A-regulated

cold and dehydration regulon (COR15A, COR15B, KIN2,

LTI78 and ERD10; Tables 2 and S2) which has lowered

expression in rcd1. However, using mutants to dissect the

direct and indirect effects of the loss of RCD1 function is

complicated due to the adjustment of plant metabolism to

the mutation; the observed gene expression response could

also reflect secondary responses. Finding the direct function

of RCD1 will require transgenic plants with inducible

expression of RCD1 combined with biochemical assay(s) of

putative RCD1 activities, for example ADP-ribosylation, and

protein stability of RCD1 and/or its interacting proteins.

Of particular interest among the genes with very high

expression in rcd1 is UPOX (At2g21640; Table 2). This gene

is considered to be a marker transcript for oxidative stress

and is induced by virtually all ROS treatments (Gadjev et al.,

2006). The high UPOX expression in non-stressed rcd1

plants is a strong indication that the absence of RCD1 alters

signaling pathways related to oxidative stress and could

contribute to ozone and paraquat phenotypes of rcd1. UPOX

encodes a small mitochondrially localized protein of

unknown function. Another gene with high expression in

rcd1 is AOX1a, encoding a mitochondrial ALTERNATIVE

OXIDASE. AOX1a activity is proposed to prevent mitochon-

drial ROS accumulation by direct transfer of electrons from

the ubiquinone pool to oxygen. Paradoxically, overexpres-

sion of AOX in tobacco resulted in increased ozone sensi-

tivity (Pasqualini et al., 2007). It is possible that the high

AOX1a expression in rcd1 could prime the plant for ozone

sensitivity.

Gene ontology analysis of genes with altered expression in

rcd1 indicated enrichment for genes involved in the apoplast

and extracellular processes, consistent with a role for RCD1 in

responses to apoplastic ROS production (Overmyer et al.,

2000). Another biological process enriched in the GO analysis

was the ubiquitin-proteasome pathway (Table S3). This

suggests that RCD1-interacting proteins, or RCD1 itself,

may be regulated at the level of protein degradation, which

could offer an explanation for the difficulty of detecting RCD1

in plant extracts even when the transgenically expressed

protein fully complements the mutant phenotype (Fig-

ure 4d,e). Interestingly, several RCD1-interacting proteins

are known or predicted to be regulated at the protein stability

level (Figure 6), such as DREB2A (Qin et al., 2008), TGA2

(Manzano et al., 2008), STO (Indorf et al., 2007), PIF3 (Bauer

et al., 2004) and PIF5 (Shen et al., 2007). This further supports

the idea that RCD1 and its targets are regulated at the protein

stability level by the proteasome pathway.

In addition to identifying the interaction partners of RCD1,

the yeast two-hybrid studies presented here also demon-

strated that the C-terminal region of RCD1 and SRO1 is

STM KNA T1 KNA T2

AS1 AS2

Organ development

ANAC046

COL10 ANAC082

AtbHLH01 1

AtbHLH019

bZIP

WRKY47 GT2-related

AtIDD5

Unknown

DREB2A

DREB2B

DREB2C

Salt/heat/osmotic stress

SOS1

DRIP1

DRIP2

ANAC013

UV -B responses

Light responses

COL9

PIF5

PIF3

ST O

phyA

phyB

PIF7 UNE10

FT , CO

Flowering ILR3

Metal homeostasis

Hormones and defense

SA

AtMYC2

JA

ABA TGA2

NPR1

IAA1 1

ET

ROS Tolerance

NPR3 NPR4

PR gene expression

JAZ, JAI3

R R C C D D 1 1

Figure 6. Summary of RADICAL-INDUCED CELL

DEATH1 (RCD1)-interacting transcription factors

(TFs) and their functions.

A simple line indicates protein–protein interac-

tion; arrows indicate positive and negative

effects on the process. Proteins known to be

regulated on the level of protein stability are

underlined.

276 Pinja Jaspers et al.

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 268–279

required for these interactions. Accordingly, analysis of rcd1

and sro1 mutants provides evidence for the importance of

the C-terminus: all mutant alleles of rcd1 have identical

phenotypes regardless of whether they are full transcript

nulls, such as rcd1-4, or if they only lack the C-terminus, like

rcd1-1, rcd1-2 and rcd1-3 (Figure S3). This indicates that the

C-terminus is crucial for interaction with TFs and RCD1

function. The same holds for the sro1 mutant, which has an

insertion site close to the C-terminus and is only truncated

by approximately 66 amino acids (Figures 1 and S3).

Although this mutant has only subtle phenotypes in the

wild-type Col-0 background, its dramatic phenotype in the

rcd1 sro1 double mutant indicates a requirement for the

C-terminus in SRO1 protein function.

A novel conserved domain was identified in the C-

terminal region of RCD1 and SRO1 and is here named the

RST domain. The RST domain is also present in the SRO2–

SRO5 (not shown) and TAF4 proteins but in no other

plant proteins. The requirement for the RCD1 and SRO1

C-terminal regions for TF interaction suggests that these

conserved sequences represent a protein–protein interac-

tion domain. The RST domain is plant-specific, as are many

of the TFs whose interaction with RCD1 requires the RST-

containing C-terminus. The presence of this putative TF-

binding domain in TAF4 suggests that the RST domain may

also be involved (with currently unknown mechanisms) in

transcriptional regulation involving the RCD1-interacting

TFs. Further experimentation is required to clarify the role

of the RST domain.

Future perspectives

RCD1 and SRO1 exemplify an important genetic principle –

unequal genetic redundancy – where one protein (RCD1) has

the major activity and its homologous partner (SRO1) plays

a minor role. The ability of RCD1 and to a lesser extent SRO1

to interact with TFs (Figure 6) can explain many of the rcd1

phenotypes. They both require their C-terminal TF-binding

domain for their activities. This C-terminus contains a novel

plant-specific conserved domain which might mediate the

protein–protein interactions. The biochemical function of

these proteins remains unknown, and the next priority

should be to verify whether these proteins have ADP

ribosylation activity and if they regulate the function of the

interacting TFs through, for example, protein stability and

proteasome-mediated protein degradation or by acting as a

coactivator affecting chromatin access or binding of the

interacting TFs.

EXPERIMENTAL PROCEDURES

Plant material and culture, construction of transgenic plants

Seeds were sown on 1:1 peat–vermiculite mixture, stratified for2 days and grown in controlled environment chambers (WeissBio1300; Weiss Gallenkamp, http://www.weiss-gallenkamp.com/)with 12-h/12-h day/night cycle, temperature 22�C/19�C, relative

humidity 70%/90%. All experiments were performed with 3-week-old plants unless otherwise stated. T-DNA insertion lines for rcd1-3(SALK_116432), rcd1-4 (GABI_229D11) and sro1-1 (SALK_074525)were obtained from Nottingham Arabidopsis Stock Centre (http://arabidopsis.info/) and the insertions were verified with the primerslisted in Table S5. Double mutants were generated by crossing sro1and rcd1-3 and genotyped in the F2 generation with insertion-specific primers. Molecular cloning procedures and generation oftransgenic plants are described in Appendix S1.

Yeast two-hybrid screens

Construction of the yeast two-hybrid library is described inAppendix S1. Yeast transformations and screens were conductedaccording to the HybridHunter manual (Invitrogen, http://www.in-vitrogen.com/) using the L40 yeast strain and LexA DBD fusions ofRCD1 and SRO1 in pHybLex as bait. For each screen, approximately200 000–500 000 colony-forming units (c.f.u.) of tryptophan auxo-trophic yeast was plated on –His selection, in the case of RCD1supplemented with 10 mM 3-aminotriazole (3AT; Sigma-Aldrich,http://www.sigmaaldrich.com/) to remove autoactivation. Colonieswere picked after 4 days of growth at 28�C and tested for b-galac-tosidase activity according to the HybridHunter manual. Putativeinteraction partners in the pYESTrp2 library plasmid were identifiedby sequencing. For confirmation, plasmids were transformed backinto yeast and assayed again for His auxotrophy and b-galactosi-dase activity. Empty vectors were used as controls for the selectionconditions.

REGIA screen

The screening of the REGIA collection was done following proce-dures described by de Folter et al. (2005), except that only one DBDconstruct at a time was tested against the whole REGIA collection. Inshort, the RCD1 or SRO1 GAL4 DBD fusion constructs in pDEST32were transformed to PJ69-4a (MATa) and mated with the matingtype MATA yeast containing the REGIA collection in pDEST22(GAL4 AD fusion) on 15 individual plates in 12 · 8 grid format(exemplified in Figure S4). After selection for the presence of bothvectors, the interaction test was done at 20�C on SD –Trp –Leu –His(+10 mM 3AT for RCD1) and SD –Trp –Leu –Ade. The growth of yeastwas followed for up to 6 days and positive colonies were subjectedto b-galactosidase assay. The interaction test was done twice forboth proteins and only those putative interaction partners that werepositive in both screens, for both histidine and adenine auxotrophy,as well as for b-galactosidase assay were selected for analysis.

The results were verified by picking the interaction partners foundin the interaction tests from the REGIA Escherichia coli entry vectorcollection in pDONR201, purifying the plasmids and sequencingthe insert. Inserts were then transferred again to pDEST22 and theinteraction test was performed with the same conditions as theoriginal screen.

GUS staining

RCD1 and SRO1 promoter activity was assessed from plants trans-formed with promoter::UidA fusions prepared as detailed inAppendix S1. The GUS staining of 14-day-old plants was doneaccording to Weigel and Glazebrook (2002). Leaf and root samplesfor cross-sectioning were embedded in plastic using the Leica His-toresin Embedding Kit according to the manufacturer’s protocol(Leica Microsystems, http://www.leica-microsystems.com/). Plantswere oriented using Ben Scheres’ oriented sections method (http://www.bio.uu.nl/mg/pd/). The GUS staining was visualized usingLeica MZ10F and Leica DML microscopes equipped with a LeicaDFC490 camera.

RCD1 and SRO1 redundancy 277

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 60, 268–279

Fluorescently tagged expression constructs

Constitutively expressed RCD1 and SRO1 YFP fusions were con-structed as detailed in Appendix S1. The plasmid was coated onto1.6 lm gold (Bio-Rad, http://www.bio-rad.com/) and bombardedinto onion epidermal cells with a Helios Gene gun (Bio-Rad). Thepeels were floated on ½ MS medium at room temperature (27�C) inthe dark for 12 h and fluorescence was observed a with Leica TCSSP 5 confocal microscope. Images were analyzed using BitplaneImaris 6.2 (http://www.bitplane.com/).

Real time quantitative RT-PCR

The RNA was isolated according to Carpenter and Simon (1998) andtreated with DNase I (MBI Fermentas, http://www.fermentas.com/).Real-time quantitative PCR was performed as previously describedwith the primers listed in Table S5 (Ahlfors et al., 2009).

Microarray experiments

Four biological replicates were used from Col-0, rcd1-1, rcd1-3, rcd1-4 and sro1. To avoid introducing bias, dye labeling was reversed intwo of the four individual hybridizations per mutant. Probe labelingand hybridization protocols and raw data files are available fromthe ArrayExpress database (http://www.ebi.ac.uk/microarray-as/ae/,accession E-MEXP-1949). The detailed procedure for data analysis ispresented in Appendix S1.

Analysis of flowering time

Plants were germinated as described above and 12 individuals ofeach accession were transferred on the same growth medium to5 · 5 cm pots, one plant per pot, and placed randomly in thechamber. Plant positions were randomized every 3 days to mini-mize effects of chamber position. The plants were monitored dailyand the day of bud emergence was recorded.

Paraquat tolerance

Leaf disks were cut from 4-week-old soil-grown plants (eightdisks/genotype) and floated on distilled water containing 1.0 lM

paraquat (methyl viologen; Sigma-Aldrich) under low light(<100 lmol m)2 sec)1) for 30 h. Chlorophyll a retention was used asthe measure of paraquat tolerance: pigments were extracted with80% acetone containing 0.01% (w/v) MgCO3 and the absorbanceat 663 nm was used to indicate the chlorophyll a content of theleaf disk. The experiment was repeated three times with similarresults.

Western hybridization

Proteins were extracted by grinding 100 mg of frozen seedlings(7 days old) with sea sand in 100 ll of protein extraction buffer(Jonak et al., 1996) supplemented with 50 lM MG132. Thirty to50 lg of total proteins was separated by 10% SDS–PAGE andtransferred into PVDF membranes (Bio-Rad). The membranes wereblocked in 5% milk powder in 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS)-saline buffer and probed with RCD1-specificantibody. Horseradish peroxidase-conjugated donkey anti-rabbitIgG (GE Healthcare, http://www6.gelifesciences.com/) was used as asecondary antibody and the signal was visualized by SuperSignalWest Pico enhanced luminescence reagents (Pierce, now part ofThermoFisher Scientific, http://www.piercenet.com/).

RCD1-specific antibody was raised in rabbit against a peptideQNQPKSKEIPGS corresponding to residues 567–578 of RCD1(Sigma-Genosys). The antibody was purified using proteinA-Sepharose (GE Healthcare). The sensitivity of the antibody was

tested by dilution series of recombinant His-tagged proteinproduced in E. coli. Additional antibodies used are described inAppendix S1.

ACKNOWLEDGEMENTS

Airi Lamminmaki, Tuomas Puukko and Marjukka Uuskallio areacknowledged for excellent technical assistance and Leena Laaksoand Pekka Lonnqvist for plant care. The funding sources acknowl-edged for this work are: Finnish Academy Centre of Excellenceprogram (2006–2011) and research grant (decision no. 121576) toJK, post-doctoral grants to KO (decision no. 115034) and MB (deci-sion no. 108760). PJ and TB were supported by the Viikki GraduateSchool in Biosciences (VGSB). RI and GA acknowledge the Neth-erlands Proteomics Centre (NPC). The REGIA TF ORF Library wasgenerated as part of the EU-funded project REGIA (http://cor-dis.europa.eu/; QLG-CT11999-00876). We acknowledge the nationalFinnish DNA Microarray Centre (Turku Centre for Biotechnology) forthe manufacturing of microarray slides and Tsuyoshi Nakagawa forproviding the pGWB13 vector.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. RADICAL-INDUCED CELL DEATH1 (RCD1) interacts invitro with four representative transcription factors.Figure S2. RADICAL-INDUCED CELL DEATH1 (RCD1) and SIMILARTO RCD-ONE1 (SRO1) subcellular localization.Figure S3. Transcripts present in mutants. Quantitative-PCR analysisof RCD1 and SRO1 transcript levels in rcd1 and sro1 mutants.Figure S4. Example plates from the REGIA screen.Table S1. Phenotypes of rcd1 and sro1 mutants. Phenotypes of rcd1and sro1 reported from the literature and from this study.Table S2. Genes with altered expression in rcd1. Microarray analysisof rcd1 compared to wild-type Col-0.Table S3. Enriched gene ontology (GO) classes in microarray data.Table S4. Additional gene expression data for promoter swapexperiment. Quantitative-PCR analysis of selected marker genes inCol-0, rcd1, sro1 and complementation lines.Table S5. Primers used in this study.Appendix S1. Supplementary experimental procedures.Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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