Biochem. J. (2012) 442, 573–581 (Printed in Great Britain)
573
doi:10.1042/BJ20111739
RCD1–DREB2A interaction in leaf senescence and stress responses in
Arabidopsis thaliana
Julia P. VAINONEN*1 , Pinja JASPERS*1 , Michael WRZACZEK*, Airi LAMMINMÄKI*, Ramesha A. REDDY*2 , Lauri VAAHTERA*,
Mikael BROSCHÉ*† and Jaakko KANGASJÄRVI*3
*Plant Biology, Department of Biosciences, Viikki Biocenter, University of Helsinki, P.O. Box 65, 00014 Helsinki, Finland, and †Institute of Technology, University of Tartu, Nooruse 1,
Tartu 50411, Estonia
Transcriptional regulation of gene expression is one major determinant of developmental control and stress adaptation in virtually all
living organisms. In recent years numerous transcription factors
controlling various aspects of plant life have been identified.
The activity of transcription factors needs to be regulated to
prevent unspecific, prolonged or inappropriate responses. The
transcription factor DREB2A (DEHYDRATION-RESPONSIVE
ELEMENT BINDING 2A) has been identified as one of the main
regulators of drought and heat responses, and it is regulated
through protein stability. In the present paper we describe
evidence that the interaction with RCD1 (RADICAL-INDUCED
CELL DEATH 1) contributes to the control of DREB2A under
a range of conditions. The interaction is mediated by a novel
protein motif in DREB2A and a splice variant of DREB2A which
lacks the interaction domain accumulates during heat stress and
senescence. In addition RCD1 is rapidly degraded during heat
stress, thus our results suggest that removal of RCD1 protein
or the loss of the interaction domain in DREB2A appears to be
required for proper DREB2A function under stress conditions.
INTRODUCTION
regulated by protein degradation via its NRD (negative regulatory
domain). NRD is required for DREB2A interaction with RING E3
ligases, and deletion of this region leads to DREB2A stabilization
of a constitutively active form of the protein [9]. Full-length
DREB2A can be stabilized through treatment with the proteasome
inhibitor MG-132, but also by heat shock [4,5].
RCD1 (RADICAL-INDUCED CELL DEATH 1) is involved in
variety of stress and developmental responses [10–12]. The rcd1
mutant has highly pleiotropic phenotypes in developmental and
stress responses, and more than 500 genes are differentially regulated at the transcriptional level in the mutant [11]. Among those
are a number of genes identified previously as direct DREB2A
targets [11]. However, RCD1 itself is not significantly regulated at
the transcriptional level, except in response to excess light treatment [13]. In yeast two-hybrid assays RCD1 interacts with numerous proteins, predominantly transcription factors [11,14–16].
Among those transcription factors were DREB2A and its close
relatives DREB2B and DREB2C. RCD1 belongs to a small plantspecific SRO (SIMILAR TO RCD ONE) protein family with six
members in A. thaliana [17]. The SROs are hallmarked by a
PARP [poly(ADP-ribose) polymerase]-like domain and possess a
RST [RCD1–SRO–TAF4 (TATA-box-binding protein-associated
factor 4)] protein–protein interaction domain at the C-terminus;
RCD1 and its closest paralogue SRO1 also contain an N-terminal
WWE domain predicted to be involved in protein–protein interactions in proteins involved in ubiquitinylation or ADP-ribosylation
processes. Despite the strong sequence conservation the SRO
PARP-like domain was shown to be catalytically inactive [17];
thus the biochemical role of the SROs continues to remain unclear.
Plant growth and productivity are negatively influenced by a
variety of abiotic stresses, such as drought, high salinity and high
temperature. Abiotic stress responses are regulated by complex
redox- and hormone-dependent signalling pathways, leading to
transcriptional reprogramming, and biochemical and physiological changes. Stress-induced changes in gene expression are to a
large extent regulated by transcription factors, and depending on
the definition, up to 10 % of the Arabidopsis thaliana genome encodes transcription factors [1]. Another major regulatory system
in plant development, hormone signalling and stress responses
is targeted protein degradation, exemplified by perception of the
hormones auxin and jasmonic acid by their F-box receptors TIR1
(TRANSPORT INHIBITOR RESPONSE 1) and COI1 (CORONATINE INSENSITIVE 1), which leads to rapid degradation of
the negative regulators AUX/IAA and JAZ (JASMONATE-ZIMDOMAIN) proteins through the proteasome [2].
The DREB (DEHYDRATION-RESPONSIVE ELEMENT
BINDING) protein family of transcription factors is known to
control abiotic stress responses in plants. Arabidopsis thaliana
class 2 DREBs, a subfamily consisting of eight members,
regulate drought- and salt-responsive signalling pathways [3–5].
Previously DREB2A and DREB2C were reported to mediate heatstress responses through HsfA3 (heat shock transcription factor
A3) [6,7,8]. Exposure to heat stress leads to fast and transient
induction of DREB2A, whereas drought and salt stress lead to
a gradual induction of DREB2A expression [4]. DREB2A is
not only regulated at the transcriptional level; protein levels are
Key words: abiotic stress, alternative splicing, CMIV-3 domain,
protein–protein interaction, protein stability, transcription factor.
Abbreviations used: ABA, abscisic acid; Col-0, Columbia-0; COR15a, COLD-REGULATED 15a; DREB, DEHYDRATION-RESPONSIVE ELEMENT
BINDING; ERD, EARLY-RESPONSIVE TO DEHYDRATION; HA, haemagglutinin; HsfA3, HEAT SHOCK TRANSCRIPTION FACTOR A3; HSP, heat-shock
protein; GST, glutathione transferase; His, histidine; MAP65-9, MICROTUBULI-ASSOCIATED PROTEIN 65-9; NRD, negative regulatory domain; PARP,
poly(ADP-ribose) polymerase; PCD, programmed cell death; SAG12, SENESCENCE-ASSOCIATED GENE 12; RCD1, RADICAL-INDUCED CELL DEATH
1; RIM, RCD1-interacting motif; ROS, reactive oxygen species; RST, RCD1–SRO–TAF4 (TATA-box-binding protein-associated factor 4); RT–PCR, real-time
PCR; qRT–PCR, quantitative RT–PCR; TAIR, The Arabidopsis Information Resource; TIP41, Tap42 (two A phosphatase associated protein 42)-interacting
protein; YFP, yellow fluorescent protein.
1
These authors contributed equally to this work.
2
Present address: Centre for Cellular and Molecular Biology, Hyderabad-500007, India.
3
To whom correspondence should be addressed ([email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
574
J. P. Vainonen and others
The interaction between RCD1 and DREB2A, two proteins
with important roles in plant stress responses, was suggested
already a decade ago [14]. However, the functional relevance of
this observation has not been addressed. The present study aims at
the analysis of the interaction of DREB2 transcription factors with
RCD1 and the investigation of its biological relevance for stress
and developmental responses. A novel conserved region of approximately 30 amino acids at the C-terminus of DREB2A and its
two paralogues is shown to be required for the DREB2A–RCD1
interaction. We demonstrated induction of DREB2A expression
during natural senescence and found a novel splice variant lacking
the RCD1-interacting domain which is induced during senescence
and heat-shock treatment. We showed that, despite the early
senescence phenotype of rcd1, the timing of expression of several
senescence marker genes is not affected in the mutant.
MATERIALS AND METHODS
Plant material and growth conditions
A. thaliana ecotype Col-0 (Columbia-0) was used as the wildtype. The rcd1-4 allele [TAIR (The Arabidopsis Information
Resource) polymorphism name GK-229D11] and dreb2a-1
(TAIR polymorphism name GK-379F02) were obtained from
the GABI-Kat collection [18]. Construction of transgenic lines
expressing HA (haemagglutinin)-tagged RCD1 was as described
previously [11].
Seeds were sown on a 1:1 peat/vermiculite mixture, stratified
for 2 days and grown in controlled environmental chambers
(Weiss 1300 growth cabinets) in 12h/12h day/night cycle,
temperature 22 ◦ C/19 ◦ C, and relative humidity 70 %/90 % in
the case of soil-grown plants. For stress treatments Arabidopsis
thaliana seedlings were grown on agar plates at 12h/12h
day/night cycle, temperature 22 ◦ C/19 ◦ C for 2 weeks. Salt
treatment was performed as described previously [15]. Heat
stress was performed as described previously [4]. For cold
treatment plants were transferred to an identical growth chamber
set at 4 ◦ C. ABA (abscisic acid) treatment was done as described
previously [10] with 50 μM for 4 and 12 h.
Yeast two-hybrid screens
Yeast work was performed as described previously [11,17] using
the GAL4-based ProQuest Y2H system (Invitrogen). Interaction
test was done at 20 ◦ C in SD-Trp-Leu-His medium in the
presence of 10 mM 3-aminotriazole, to eliminate autoactivation,
and SD-Trp-Leu-Ade medium. Yeast growth was monitored
for up to 6 days, and positive colonies were subjected to βgalactosidase assay. All interaction tests were performed twice
and only colonies grown on both selection plates and positive
in β-galactosidase assay were regarded as positive. The primers
used for cloning constructs for the interaction tests are described
in Supplementary Table S1 (at http://www.BiochemJ.org/bj/442/
bj4420573add.htm).
a Bio-Rad Laboratories CFX384 Real-Time PCR Detection
System. Primers used for qRT–PCR are listed in Supplementary
Table S1. Analysis of qRT–PCR results was performed using
the qBase2.1 program package (Biogazelle) [20]. Several
different reference genes were selected from [21] and verified
to have stable expression in publically available microarray
experiments (Genevestigator, https://www.genevestigator.com/).
Reference genes were further evaluated for expression stability
with geNorm (Supplementary Table S1) [22]. TIP41 [Tap42 (two
A phosphatase associated protein 42)-interacting protein] was
used for normalization of the ABA and senescence experiments;
TIP41, SAND, YLS8 and At4g33380 were used for normalization
of the heat-shock experiment. Analysis of DREB2A splice variants
was done by RT–PCR (real-time PCR) using primers specific
for DREB2A.1 and DREB2A.2 (Supplementary Table S1) and
subsequent separation on polyacrylamide gels. Amplification of
the DREB2A.1 and DREB2A.2 cDNA was performed with hot
start PCR using Maxima® Hot Start Green PCR Master Mix
(Thermo Scientific). A mixture of two plasmids carrying either
DREB2A full-length or DREB2A244–275 cDNAs was used as
a control for the specificity of PCR products.
Protein extraction and immunoblotting
Proteins were extracted by grinding frozen seedlings in RIPA
buffer [50 mM Tris/HCl (pH 8.0), 150 mM NaCl, 1 % Triton X100, 0.5 % sodium deoxycholate and 0.1 % SDS] in the presence
of protease inhibitor cocktail (1:200 dilution; Sigma), 2 mM DTT
(dithiothreitol) and 5 mM EDTA. The samples were centrifuged
at 16 000 g for 15 min at 4 ◦ C, and the supernatant was used
for Western blot analyses. Protein concentration was determined
by the Lowry method using DC Protein Assay reagents (BioRad Laboratories). Proteins were separated by SDS/PAGE and
transferred on to PVDF membranes (Bio-Rad Laboratories).
After blocking with 5 % skimmed milk powder in Tris-buffered
saline with 0.05 % Tween-20 the membranes were incubated
with protein-specific antibodies. HA–tagged RCD1 was detected
using anti-HA high-affinity monoclonal antibody (clone 3F10,
Roche) with protein loading of 100 μg per lane. HSP (heat-shock
protein) 101 was detected using anti-HSP101 polyclonal antibody
(AS07 253, Agrisera) with protein loading of 2–5 μg per lane.
Peroxidase-conjugated anti-rat IgG (Sigma) and anti-rabbit IgG
(GE Healthcare) were used as secondary antibodies; the signal
was visualized by SuperSignal West Pico luminescence reagents
(Thermo Scientific).
Protein sequence analysis
Protein structure predictions for the RST domain of RCD1 and
for the C-terminal domain of DREB2A were done using the ITASSER server [23] at http://zhanglab.ccmb.med.umich.edu/ITASSER.
In vitro pull-down assay
RESULTS AND DISCUSSION
Expression and purification of the recombinant RCD1–His
(histidine) and DREB2A–GST (glutathione transferase) proteins
was performed as described previously [11]. The pull-down
assays were performed by incubation of DREB2A–GST proteins
immobilized on GSH beads with His-tagged RCD1 protein as
described previously [11].
A sub-motif of the CMIV-3 domain mediates interaction of DREB2
with RCD1
RNA extraction and qRT–PCR (quantitative real-time PCR)
RNA isolation and qRT–PCR experiments were performed as
described previously [19]. qRT–PCR was carried out using
c The Authors Journal compilation c 2012 Biochemical Society
A. thaliana RCD1 interacts with three out of eight members of the
DREB2 subfamily of transcription factors: DREB2A, DREB2B
and DREB2C [11]. These proteins possess a conserved sequence
motif at their C-termini named CMIV-3 [24]. Other members
of the DREB2 subfamily lacking the CMIV-3 motif, such as
DREB2E, did not interact with RCD1 (Figure 1).
Alignment of the CMIV-3 domain from DREB2A, DREB2B
and DREB2C identified a conserved stretch of amino acids with
Alternative splice variant of DREB2A lacks interaction with RCD1
Figure 1
575
RCD1 interacts with three members of the DREB2 subfamily containing the CMIV-3 domain
The presence of the CMIV-3 domain in DREB2 proteins correlates with their interaction with RCD1. Shown are the phylogenetic tree for the DREB2 subfamily, and the domain structure of the DREB2
proteins used as preys in the yeast two-hybrid interaction test with RCD1 (right-hand panel). DREB2A and DREB2B are highly similar and interact with RCD1. DREB2C shows higher similarity to
DREB2H, but interacts with RCD1 due to the presence of the CMIV-3 motif.
three clusters of strictly conserved residues FDXXELLXXLN
(X denotes any amino acid), corresponding to FDVDELLRDLN
in DREB2A; hereafter this motif will be referred to as RIM
(RCD1-interacting motif). A mutational approach in combination
with interaction testing in the yeast two-hybrid system was
used to reveal the role of the conserved motif in protein–
protein interaction. A deletion construct of DREB2A protein,
DREB2A244–275, lacking the whole CMIV-3 motif (residues
248–279 in DREB2A) was created to test if CMIV-3 is required
for the interaction with RCD1. The role of conserved amino
acids within the RIM sequence of DREB2A was addressed by
substitutions of these amino acids with alanines: F259A/D260A
(m1), E263A/L264A/L265A (m2) and L268A/N269A (m3)
(Figure 2A). All of the constructs were tested for the interaction
with RCD1 in the yeast two-hybrid system.
Deletion of the CMIV-3 motif as well as mutations
F259A/D260A and E263A/L264A/L265A completely abolished the DREB2A–RCD1 interaction, whereas the variant
L268A/N269A was still able to interact with RCD1 (Figure 2B).
Yeast two-hybrid data were verified by in vitro pull-down assays
using recombinant RCD1 protein and GST-fused DREB2A
variants captured on GSH beads. This analysis (Figure 2C)
confirmed the results of the yeast two-hybrid screens. Thus the
Figure 2
CMIV-3 domain and the conserved residues Phe259 /Asp260 and
Glu263 /Leu264 /Leu265 were required for the interaction of DREB2A
with RCD1.
The conserved sequence RIM is not sufficient to mediate
interaction of RCD1 with other proteins
Although RIM appears to be critical for RCD1 interaction
with DREB2 proteins, it was not present in other proteins
that were found to interact with RCD1 [11]. A search
in all proteins in A. thaliana using the degenerate motif
FDXXXLLXX[ILMV][END], based on the CMIV-3 box
sequences of several plant species, identified 15 proteins carrying
the motif, including DREB2A, DREB2B and DREB2C (Table 1).
Interestingly, with the exception of the three DREB family
members, none of the identified proteins has previously been
characterized as an RCD1-interacting protein and none of these
proteins was a transcription factor. Presence of the RIM motif
might be an indication of their potential interaction with RCD1.
However, the motif, due to its short length, might not be
sufficient to mediate the interaction. To address this question,
we fused the C-terminal part of DREB2A (residues 250–335) or
Conserved motifs in DREB2A and RCD1 mediating protein–protein interaction
A mutation approach and yeast two-hybrid interaction tests identify conserved elements in DREB2A and RCD1 necessary for the interaction. (A) Alignment of the CMIV-3 sequences of DREB2A,
DREB2B and DREB2C showing the conserved amino acids. Substitution of conserved residues to alanines (A) is denoted by m1, m2 and m3. (B) Yeast spots from the interaction test of DREB2A
constructs with RCD1. The 244–275 construct is a deletion of these amino acids in DREB2A. (C) Western blot of the in vitro pull-down assay. The band at 70 kDa corresponds to the recombinant
RCD1 captured by immobilized DREB2A protein variants fused to GST. GST was used as a negative control. (D) Yeast spots from the interaction test of the fusion proteins with RCD1 as bait.
DREB2A, DREB2E, YFP and MAP65-9 were used as prey constructs to test interaction with RCD1. Additional prey constructs consist of fusion proteins between parts of DREB2A with DREB2E or
YFP respectively. DREB2E–DREB2A-CT and YFP–DREB2A-CT: the C-terminus of DREB2A (residues 250–335, including the RIM) was added to DREB2E or YFP respectively. DREB2E–DREB2A-RIM
and YFP–DREB2A-RIM: the RIM of DREB2A (residues 250–277) was added to DREB2E or YFP respectively (E) Yeast spots from the interaction test of RCD1 point mutation constructs with DREB2A.
569–589 corresponds to the RCD1569–589 construct used as the shortest C-terminal truncation construct that retains the interaction. empty, an empty vector control.
c The Authors Journal compilation c 2012 Biochemical Society
576
J. P. Vainonen and others
Table 1 Arabidopsis thaliana proteins containing the FDXXXLLXX[ILMV][END] motif
AGI, Arabidopsis Genome Initiative; TF, transcription factor.
AGI code
Name
TF
At1g20480
At3g01670
At1g64110
At5g05410
At5g62250
At2g02955
At1g74310
At1g55810
At3g11020
At1g72500
At4g14670
At2g40340
At4g26510
At5g07270
At3g27190
4-coumarate-CoA ligase family protein 2
Unknown protein
DUO1-activated ATPase1
DREB2A
MICROTUBULE-ASSOCIATED PROTEIN 65-9 (MAP65-9)
MATERNAL EFFECT EMBRYO ARREST 12 (MEE12)
HEAT SHOCK PROTEIN 101 (HSP101)
Putative uracil phosphoribosyltransferase
DREB2B
Inter-α-trypsin inhibitor heavy chain-related protein
CASEIN LYTIC PROTEINASE B2 (CLPB2)
DREB2C
Uridine kinase-like protein 4
Ankyrin repeat family protein
Uridine kinase-like protein 2, chloroplastic
−
−
−
+
−
−
−
−
+
−
−
+
−
−
−
its RIM sequence alone (residues 250–277) to the C-termini of
two non-RCD1-interacting proteins, DREB2E and YFP (yellow
fluorescent protein), and tested these constructs for interaction
with RCD1 using the yeast two-hybrid assay. In addition,
MAP65-9 (MICROTUBULI-ASSOCIATED PROTEIN 65-9)
was chosen as a protein which contained the conserved motif
according to the bioinformatic analysis (Table 1), but has not
previously been identified as an RCD1-interaction partner.
Addition of the C-terminus or the shorter conserved RIM
sequence from DREB2A to DREB2E was sufficient to make
the resulting hybrid protein interact with RCD1 (Figure 2D).
However, fusion of the same RIM sequence to YFP, or the
presence of the motif in MAP65-9 was not sufficient to
enable interaction. Interestingly, addition of the C-terminus
of DREB2A (residues 250–335 including RIM) to YFP was
enough to cause interaction with RCD1 in the yeast two-hybrid
analysis. This indicates that the RIM is clearly central for the
interaction of DREBs with RCD1. Surrounding sequences are
required to form the interacting structure together with the RIM,
which is likely to be an α-helical fold according to sequence
analysis (Supplementary Figure S1 at http://www.BiochemJ.org/
bj/442/bj4420573add.htm). Other transcription factors and
proteins interacting with RCD1 do apparently not need the RIM.
The domains responsible for the interaction of those proteins with
RCD1 might be conserved at a three-dimensional structural level
rather than at the primary amino acid level. In addition, the RST
domain might be structurally flexible to allow the binding of
the different interaction domains from various RCD1-interacting
proteins.
RCD1 residues necessary for the interaction with DREB2A
After identification of the important residues in DREB2A RIM
that mediate the RCD1–DREB2A interaction, we identified
critical residues in RCD1 required for the interaction. The Cterminal RST domain of RCD1 has been shown to mediate
the interaction with transcription factors [11,17]. Alignment
of the RST domains of SRO proteins from several plant species
revealed a number of conserved amino acids within the RST
domain of RCD1, including Ser505 , Ser518 , Tyr533 and several
leucine and isoleucine residues [17]. To address the role of
these amino acids in the RCD1–DREB2A interactions, seven
variants of RCD1 with point mutations of eight conserved residues
c The Authors Journal compilation c 2012 Biochemical Society
were created (a list is provided in Supplementary Table S2 at
http://www.BiochemJ.org/bj/442/bj4420573add.htm) and tested
for interaction with DREB2A using the yeast two-hybrid assay.
Three of the eight mutated amino acids were found to
be necessary for the interaction: substitutions L528Q/I529Q
and I563Q in RCD1 completely abolished its interaction with
DREB2A (Figure 2E). The high number of conserved leucine
residues and their involvement in protein–protein interactions
points towards a possible α-helical structure of the RST domain.
This was confirmed by predictions made with the I-TASSER
server (http://zhanglab.ccmb.med.umich.edu/I-TASSER). The
leucine residues important for the RCD1–DREB2A interaction
were, with high probability, embedded within α-helices
(Supplementary Figure S1). Interestingly, the conserved amino
acid stretch ELL in DREB2A (marked as m2 on Figure 2A),
critical for the interaction, was also within an α-helix according
to the I-TASSER prediction (Supplementary Figure S1).
DREB2A protein levels are tightly regulated via the proteasome
system and RCD1 is present in very low amounts [11], which
makes in vivo verification of their interaction difficult. Therefore
we addressed the biological role of the interaction by analysis
of gene expression in response to several abiotic stresses in the
Col-0, rcd1 and dreb2a strains.
An alternative splice variant DREB2A.2 induced during senescence
and heat shock lacks RIM
DREB2A exists in two splice variants, DREB2A.1 and a
shorter DREB2A.2. Differential regulation of alternatively spliced
transcripts has been shown for DREB2 in wheat, barley and maize
[25–27], but not for A. thaliana.
Significantly, the alternative splice variant would produce
a protein which specifically lacks the CMIV-3 motif and,
consequently, the RIM required for the interaction with RCD1
(Figure 3A), and expression of this variant during stress could
be a way to regulate the RCD1–DREB2A interaction. The
splice variant lacking CMIV-3 (residues 248–279) would retain
transcriptional activation activity since a DREB2A deletion
mutant lacking residues 254–281 has been reported to show
the same transactivation of a reporter gene as the full-length
protein [5]. However, there are no data on translation of the splice
variant in planta. To address this question at the protein level we
obtained a DREB2A-specific antibody which would recognize
both variants. Unfortunately the antibody was unspecific, even
after affinity purification, and therefore not usable (results not
shown).
To get information on the expression of DREB2A.2 splice
variant we searched available cDNA sequences for the presence
of DREB2A.2. A senescence cDNA library was found to
contain a cDNA corresponding exactly to the DREB2A.2 gene
model (GenBank® accession number CD530293). Therefore the
expression of DREB2A splice variants during senescence was
investigated both in wild-type and in rcd1 plants using a set
of primers specific to each splicing variant (Figure 3A). A
dreb2a knockout mutant was used as a negative control in the
experiments. The senescence process in rcd1, dreb2a and wildtype plants was also monitored (Figure 3B).
The splice variant DREB2A.2, lacking the CMIV-3 domain, was
specifically induced during senescence (Figure 3C). DREB2A.1
expression was also induced, and, to our knowledge, this is the
first report of DREB2A induction caused by senescence. There
were no differences in the timing of DREB2A induction between
wild-type and rcd1 plants (Figure 3C), although the mutant
showed an early senescence phenotype and the dreb2a mutant
appeared to senesce normally (Figure 3B). This indicated that
Alternative splice variant of DREB2A lacks interaction with RCD1
Figure 3
577
Induction of DREB2A.2 splice variant lacking RIM during senescence and heat shock
(A) DREB2A gene model showing two splice variants and their domain structure. The primers used for RT–PCR analyses (LP1, LP2 and RP) are shown with arrows. AP2, AP2/ERF domain; NLS,
nuclear localization signal; NRD, negative regulatory domain. (B) Leaf ageing process in Col-0, rcd1 and dreb2a plants. Representative images of soil-grown plants photographed at 3, 5, 6 and
8 weeks after sowing are shown. Scale bars = 1 cm. (C) RT–PCR analysis of DREB2A splice variants expression at 3, 5, 6 and 8 weeks (wk) after sowing. (D) RT–PCR analysis of DREB2A splice
variant expression after 0, 1, 3, and 5 h of heat stress (37 ◦ C). TIP41 was used as a reference gene. Mix denotes a mixture of the DREB2A variants cDNAs used as a control for specificity, Representative
images of two to three independent experiments are shown. CDS, coding sequence; N, a negative control without a template; UTR, untranslated region.
the DREB2A induction was not directly related to the initiation
of senescence. Senescence, however, is a complicated process
involving ROS (reactive oxygen species) at the signalling level
and death and drying of tissues at the physiological level. The
DREB2A.2 splice variant might well play a role in one of
the many senescence-specific signalling cascades. It has been
shown that expression of alternatively spliced transcripts may
change at different developmental stages as well as in response to
environmental changes [28,29].
The expression of DREB2A splice variants was investigated
under abiotic stresses known to induce DREB2A expression
including salt, cold and heat stresses. As expected, salt
and cold treatments resulted in induction of DREB2A.1
(Supplementary Figure S2 at http://www.BiochemJ.org/bj/442/
bj4420573add.htm). No increase in the transcript level was
detected for DREB2A.2 under these conditions. In contrast, heat
stress resulted in clear and fast induction of both splice variants
of DREB2A (Figure 3D). Rapid induction of DREB2A.1 caused
by heat stress has been described previously [4], whereas the
induction of the DREB2A.2 transcript under heat stress is a
novel finding. Expression of TIP41 used as a reference gene was
significantly decreased under heat stress (Figure 3D), suggesting
that the relative induction of DREB2A transcripts was even higher.
We have tested two other reference genes with the same results
(results not shown; see more below about reference gene stability
during heat shock).
The rcd1 mutant displays an unusual type of senescence
To characterize the early senescence phenotype of rcd1 and
confirm the normal senescence pattern of dreb2a (Figure 3B)
at the molecular level we monitored expression of senescence
marker genes by qRT–PCR in the mutants and wild-type plants
during natural senescence. Marker genes for qRT–PCR were
selected from senescence-regulated genes described previously
[30], and show contrasting expression patterns during senescence
(i.e. reduced expression and early or late induced expression).
Furthermore, SAG12 (SENESCENCE-ASSOCIATED GENE 12)
was included since it is considered to be a senescence-specific
marker gene [31].
According to the qRT–PCR data (Figure 4) all five selected
genes showed similar expression patterns in dreb2a as in Col-0
plants, which agrees with the normal senescence phenotype of
the mutant. In the rcd1 mutant the expression of ERD (EARLYRESPONSIVE TO DEHYDRATION) 3, ERD10, SAG12 and
ZAT10 was the same as in Col-0 plants. However, expression
of NFXL1 was higher in rcd1 than in Col-0 plants already
at the earliest time point and increased with time. NFXL1
has been shown to be a target of DREB2A using plants
overexpressing constitutively active DREB2A [4]. The increased
NFXL1 expression in the rcd1 mutant is thus consistent with a
model where RCD1 would negatively regulate DREB2A levels,
and consequently rcd1 plants would have increased amounts of
DREB2A resulting in higher NFXL1 expression.
Since the senescence process in the dreb2a mutants was unaltered (Figure 3B), and senescence gene expression was normal
in dreb2a plants, it seems that absence of DREB2A does
not affect the senescence process, possibly due to redundancy
between DREB2A, DREB2B and DREB2C. A more definitive
proof for the involvement of DREB2A in senescence could be
obtained from analysis of plants overexpressing constitutively
active DREB2A.
c The Authors Journal compilation c 2012 Biochemical Society
578
Figure 4
J. P. Vainonen and others
Unaltered expression of senescence marker genes in the early-senescing rcd1 mutant
Senescence marker genes show similar expression patterns in Col-0, dreb2a and rcd1 plants. Only one marker, NFXL1, showed higher expression in rcd1 plants upon senescence. Transcript levels
of selected genes were investigated by qRT–PCR. Data represent means and standard errors of three biological replicates.
The accelerated visible senescence of the rcd1 mutant,
combined with normal expression of the senescence-specific
marker SAG12, indicates that senescence in rcd1 plants differs
from the normal ageing process, and might represent another
type of tissue death. The hallmark phenotype of rcd1mutants is
altered PCD (programmed cell death) in response to apoplastic
ROS (reactive oxygen species) treatments [32], and PCD is a
part of the senescence process [31]. Thus what appears to the
macroscopic observation as early senescence in rcd1 plants could
instead be the long-term consequence of the plant having altered
PCD due to the lack of functional RCD1. The rcd1 mutant offers
an attractive complement to other early and late senescing mutants
in the further study of regulation of senescence [31,33,34].
Heat-stress responses are pre-activated in rcd1 plants
Interestingly, the gene expression profile of the rcd1 mutant
in control conditions shared some similarities with the gene
expression profile of wild-type plants after 2 h of heat shock
(T. Blomster, U. Brosché, J. Salojärvi, N. Sipari, A. Lamminmäki,
F. Cui, S. Narayanasamy, R. A. Reddy, U. Keinänen, K. Overmyer
and J. Kangasjärvi, unpublished work). In order to compare the
heat-stress response of wild-type with the rcd1 and dreb2a mutants
we performed qRT–PCR analysis of known heat-stress marker
genes. RCD1 was also included in the analysis since RCD1 might
play a role in the heat shock response. All of the analysed marker
genes, with the exception of RCD1, showed clearly elevated
transcript levels after application of heat stress at 37 ◦ C for 1,
3 or 5 h respectively (Figure 5).
DREB2A expression was elevated after heat stress in wild-type
and rcd1 plants; the transcript was highly reduced in the dreb2a
c The Authors Journal compilation c 2012 Biochemical Society
mutant. RCD1 showed approximately two-fold reduction in
transcript levels after heat stress (Figure 6). Transcript levels were
slightly higher in dreb2a plants under control conditions compared
with the wild-type. Analysis of other heat-shock responsive genes
showed only slight differences in their expression among the
genotypes (Figure 5). CYP18-1 showed higher expression levels in
the rcd1 mutant in response to heat stress compared with the wildtype and dreb2a plants, whereas the expression of an HSP20-like
chaperone was slightly higher in dreb2a compared with wild-type
and rcd1 plants in response to heat stress. Heat-stress induction of
transcript levels of HSP101 showed a slight delay in dreb2a and
rcd1 compared with wild-type plants, whereas HSP70 transcript
levels were reduced in the mutants compared with the wild-type.
Conversely HSP18.2 displayed a slightly earlier induction after
heat stress in dreb2a compared with the wild-type and rcd1 plants.
According to Sakuma et al. [4], the induction of HSP70,
HSP18.2 and CYP18-1 under heat stress should be significantly
lower in dreb2a compared with Col-0 plants, which was not seen
in our experimental conditions. The discrepancy in the data might
be explained by possible differences in the experimental setup,
for example, different light source and intensity. It has also been
shown that DREB2A is not a direct activator of HSP genes,
which are instead activated by HsfA3 [7]. In turn, HsfA3 could
be activated by not only DREB2A, but also by DREB2B and
DREB2C, and a double or triple mutant might have a stronger
phenotype [6,7].
In addition to activation of HSP genes, the heat stress led
to a general decreased expression of genes with housekeeping
function. Accurate gene expression analysis using qRT–PCR
require the use of reference genes (also known as housekeeping
genes) for normalization that exhibit stable expression in the
Alternative splice variant of DREB2A lacks interaction with RCD1
Figure 5
579
Induction of heat-responsive genes is largely unaltered in rcd1 plants
Transcript levels of heat-responsive genes in Col-0, rcd1 and dreb2a plants under heat shock were determined by qRT–PCR. Only CYP18-1 shows higher induction in the rcd1 mutant. Data represent
means and standard errors of two biological replicates; data is plotted on a log10 scale.
Figure 6
RCD1 protein is quickly degraded after heat stress
The protein content in Col-0, rcd1 and dreb2a plants was monitored by Western blot analyses.
(A) Accumulation of HSP101 under heat shock (indicated hours at 37 ◦ C). Per lane, 2 μg
of total protein was loaded. (B) Steady-state HSP101 levels in unstressed control plants. Per
lane, 5 μg of total protein was loaded. (C) Degradation of HA-tagged RCD1 during heat-shock
response. Per lane, 100 μg of total protein was loaded. Arrow indicates unspecific signal.
Rubisco large subunit (RbcL) amounts are shown in all panels as a control for equal protein
loading. Representative images of two independent experiments are shown.
samples to be analysed [21,35]. We tested several reference genes
which were selected from [21] and confirmed to have stable
expression on the basis of available heat stress microarray data
(https://www.genevestigator.com/gv/). Despite this, all reference
genes showed higher (3–5×) Ct values in the heat-stressed
samples compared to controls (Supplementary Table S1). A
higher Ct value corresponds to lower expression since more
PCR cycles are required to reach the cycle threshold. Normal
RT–PCR also showed reduced expression of the reference gene
TIP41 in response to heat shock (Figure 3D). To enable accurate
normalization of the heat shock qRT–PCR data we used four
reference genes for normalization, that displayed the best geNorm
stability values, and this design allowed us to obtain reliable
results from qRT–PCR analysis (Supplementary Table S1).
This suggests that many previously reported qRT–PCR datasets
for heat stress probably suffer from unsuitable reference gene
selection and should be re-analysed using established guidelines
and algorithms for qRT–PCR analysis [35,36], especially if
Actin-2 has been used for normalization since this gene had by
far the worst stability value.
To verify the response to heat stress on the protein level we
monitored the level of HSP101 protein in rcd1, dreb2a and
Col-0 plants under heat stress by Western blot analyses. As
described previously, heat stress led to significant accumulation
of HSP101 within 1 h of the treatment. Notably, the level of
HSP101 was higher in untreated rcd1 than in Col-0 plants
(Figures 6A and 6B; note that more protein is loaded in 6B).
This result is in line with our unpublished data on the gene
expression profile of control-grown rcd1plants being reminiscent
of heat-stressed wild type plants (T. Blomster, U. Brosché, J.
Salojärvi, N. Sipari, A. Lamminmäki, F. Cui, S. Narayanasamy,
R. A. Reddy, U. Keinänen, K. Overmyer and J. Kangasjärvi,
unpublished work). To address the role of RCD1 in the heatshock response we monitored the protein level under stress
treatment. Since the available RCD1-specific antibodies were
not sensitive enough to detect the native protein [11] (results
not shown), we used transgenic lines expressing HA-tagged
RCD1 under the control of its native promoter. Application
of heat stress resulted in fast degradation of RCD1 within
30 min, whereas the protein level in the control samples remained
the same (Figure 6C). Significantly, RCD1 down-regulation
at the protein level was much stronger than the reduction of
the corresponding transcript level (Figure 5). This confirms the
observation that changes in protein expression are not always
correlated with the changes in transcript level [37,38].
The RCD1 protein amounts were significantly reduced after
30 min of heat shock, whereas DREB2A was shown to be
stabilized after 1 h of heat shock. This opposite direction of protein
accumulation suggests the negative impact of RCD1–DREB2A
interaction, or in other words, RCD1 could be involved in targeting
DREB2A for degradation. The induction of DREB2A.2 lacking
c The Authors Journal compilation c 2012 Biochemical Society
580
Figure 7
J. P. Vainonen and others
Unaltered ABA gene expression responses in rcd1 plants
Col-0, rcd1 and dreb2a plants were treated with 50 μm ABA and the transcript levels of three ABA marker genes, COR15a , EARLI-family gene1 and KIN2 , were investigated after 4 and 12 h of
treatment by qRT–PCR. Data represent means and standard errors of three biological replicates.
RIM under heat treatment might then serve as a complementary or
safeguard mechanism to avoid the RCD1–DREB2A interaction.
The increased expression of the DREB2A.2 splice variant during
senescence points towards similar mechanisms at play during the
heat response and senescence. However, this hypothesis requires
additional verification.
ABA-regulated gene expression is normal in rcd1 and dreb2a plants
Abiotic stresses such as salt, cold and drought regulate gene
expression via ABA-dependent and -independent signalling
pathways [39]. We have shown previously that the rcd1 mutant
is ABA insensitive based on reduced ABA activation of the
ABA-induced gene RAB18 (RESPONSIVE TO ABA 18) [10]. In
contrast, DREB2A is located on the ABA-independent signalling
pathway [39]. To elucidate whether RCD1 and DREB2A interact
in the ABA signalling pathway, Col-0, rcd1 and dreb2a plants
were treated with ABA and the samples harvested after 4 and 12 h.
Gene expression was analysed with qRT–PCR. ABA effectively
increased expression of COR15a (COLD-REGULATED 15a)
and KIN2, and reduced expression of the EARLI1 (EARLY
ARABIDOPSIS ALUMINUM-INDUCED 1) family gene. No
major differences were found between the mutants and Col-0
(Figure 7), indicating that the rcd1mutant is not generally ABA
insensitive and that RCD1–DREB2A is probably not acting in
ABA signalling.
analysis of the mechanism of RCD1 regulation. Despite visually
accelerated senescence in rcd1 plants, the mutant had normal
expression of senescence related genes, suggesting that the early
senescence in the rcd1 mutant is an atypical process. This makes
the rcd1 mutant a new tool for dissecting regulatory mechanisms
of senescence in A. thaliana. RCD1 appears to be regulated at the
protein level in response to stress (results not shown). Our analysis
provides conclusive evidence for direct interaction of RCD1 and
DREB2 transcription factors (DREB2A, B and C) in vitro and
supports the existence of an RCD1–DREB2A regulatory unit
controlling abiotic stress responses in A. thaliana. Future analysis
of the in vivo interactions between the two proteins will allow
exciting insights into transcription factor regulation and help to
answer some of the many questions surrounding the co-ordination
of transcriptional responses to environmental stimuli.
AUTHOR CONTRIBUTION
Julia Vainonen, Pinja Jaspers, Michael Wrzaczek, Airi Lamminmäki, Ramesha Reddy, Lauri
Vaahtera and Mikael Brosché conducted the experiments; Julia Vainonen, Pinja Jaspers,
Michael Wrzaczek, Mikael Brosché and Jaakko Kangasjärvi designed the experiments and
analysed the data; and Julia Vainonen, Pinja Jaspers, Michael Wrzaczek, Mikael Brosché
and Jaakko Kangasjärvi prepared the paper.
ACKNOWLEDGEMENTS
RCD1 mediates control of DREB2A through protein stability under
stress conditions
Protein stability and targeted protein degradation are major
regulatory processes in A. thaliana exemplified by, for example,
a large number genes encoding components of the ubiquitin–
proteasome pathway [40]. During heat shock, RCD1 and
DREB2A were inversely regulated, i.e. RCD1 was degraded
and DREB2A was stabilized (Figure 7 and [4]). Several lines
of evidence suggest that the removal of RCD1 is required for
proper DREB2A function: (i) RCD1 interacts with DREB2A
through a specific RIM sequence; (ii) during heat shock and
senescence, an alternatively spliced variant of DREB2A is formed
which leads to the formation of a protein lacking RIM and
therefore cannot interact with RCD1; and (iii) NFXL1, a target of
DREB2A, accumulates to higher levels in the rcd1 mutant. The
study of DREB2A is complicated by very low protein levels and
redundancy between its close paralogues DREB2B and DREB2C,
thus the use of double and triple mutants would help to clarify
the role of these transcription factors in stress and senescence.
Similarly, the low protein abundance of RCD1 complicates
c The Authors Journal compilation c 2012 Biochemical Society
We thank Dr Jorma Vahala for helpful comments and suggestions on fusion protein design.
We thank Tuomas Puukko and Marjukka Uuskallio for excellent technical assistance and
Leena Grönholm for taking care of plants. We thank Dr Franziska Turck (Max Planck Institute
for Plant Breeding Research, Cologne, Germany) for providing yeast strains containing
the REGIA TF ORF Library, generated as part of the European Union-funded project
REGIA. Professor Gerco Angenent and Dr Richard Immink (Plant Research International
Wageningen, The Netherlands) are gratefully acknowledged for their collaboration in the
yeast two-hybrid work.
FUNDING
This work was supported by the University of Helsinki, Academy of Finland and the Viikki
Graduate School in Biosciences.
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Received 28 September 2011/5 December 2011; accepted 12 December 2011
Published as BJ Immediate Publication 12 December 2011, doi:10.1042/BJ20111739
c The Authors Journal compilation c 2012 Biochemical Society
Biochem. J. (2012) 442, 573–581 (Printed in Great Britain)
doi:10.1042/BJ20111739
SUPPLEMENTARY ONLINE DATA
RCD1–DREB2A interaction in leaf senescence and stress responses in
Arabidopsis thaliana
Julia P. VAINONEN*1 , Pinja JASPERS*1 , Michael WRZACZEK*, Airi LAMMINMÄKI*, Ramesha A. REDDY*2 , Lauri VAAHTERA*,
Mikael BROSCHÉ*† and Jaakko KANGASJÄRVI*3
*Plant Biology, Department of Biosciences, Viikki Biocenter, University of Helsinki, P.O. Box 65, 00014 Helsinki, Finland, and †Institute of Technology, University of Tartu, Nooruse 1,
Tartu 50411, Estonia
Figure S1
Secondary structures of the RST domain of RCD1 and the C-terminal domain of DREB2A
Prediction of the secondary structure elements was done by I-TASSER. Red colour indicates amino acids necessary for the RCD1–DREB2A interaction. Positions of these residues are shown above
the protein sequences.
Figure S2
Induction of DREB2A.1 during salt and cold stresses
Col-0 seedlings were either treated with 250 mM NaCl (upper panel) or incubated at 4◦ C (lower
panel). Expression of DREB2A splice variants was analysed by RT–PCR using specific primers.
Mix, mixture of the DREB2A variants cDNAs used as a control for primer specificity; N1 and N2,
negative controls without a template and with the cDNA from dreb2a respectively.
1
2
3
These authors contributed equally to this work.
Present address: Centre for Cellular and Molecular Biology, Hyderabad-500007, India.
To whom correspondence should be addressed ([email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
J. P. Vainonen and others
Table S1 Primers used in the present study, reference gene stability
determined with geNorm and Ct values of reference genes in the heat shock
qRT–PCR experiment
Available at http://www.biochemj.org/bj/442/bj4420573add.htm.
Table S2 Effect of the point mutations within the RST domain of RCD1 on
the RCD1–DREB2A interaction
RCD1 mutation
Interaction with DREB2A
S505D
S518D
L528Q/I529Q
Y533F
T542D
L558Q
I563Q
+
+
−
+
+
+
−
Received 28 September 2011/5 December 2011; accepted 12 December 2011
Published as BJ Immediate Publication 12 December 2011, doi:10.1042/BJ20111739
c The Authors Journal compilation c 2012 Biochemical Society