Research
COP1 promotes ABA-induced stomatal closure by modulating
the abundance of ABI/HAB and AHG3 phosphatases
Qingbin Chen1* , Ling Bai1* , Wenjing Wang1* , Huazhong Shi2
Kang Liu1, Hong-Quan Yang4 and Chun-Peng Song1
, Jose Ram
on Botella3
, Qidi Zhan1,
1
State Key Laboratory of Crop Stress Adaptation and Improvement, School of Life Sciences, Henan University, 85 Minglun Street, Kaifeng 475001, China; 2Department of Chemistry and
Biochemistry, Texas Tech University, Lubbock, TX 79409, USA; 3Plant Genetic Engineering Laboratory, School of Agriculture and Food Sciences, The University of Queensland, Brisbane,
Queensland 4072, Australia; 4Shanghai Key Laboratory of Plant Molecular Sciences, College of Life Sciences, Shanghai Normal University, Shanghai 200234, China
Summary
Author for correspondence:
Chun-Peng Song
Email: songcp@henu.edu.cn
Received: 15 March 2020
Accepted: 1 October 2020
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doi: 10.1111/nph.17001
Key words: abscisic acid (ABA), clade A
PP2C, COP1, stomatal movement,
ubiquitination.
Plant stomata play a crucial role in leaf function, controlling water transpiration in response
to environmental stresses and modulating the gas exchange necessary for photosynthesis.
The phytohormone abscisic acid (ABA) promotes stomatal closure and inhibits light-induced
stomatal opening. The Arabidopsis thaliana E3 ubiquitin ligase COP1 functions in ABA-mediated stomatal closure. However, the underlying molecular mechanisms are still not fully
understood.
Yeast two-hybrid assays were used to identify ABA signaling components that interact with
COP1, and biochemical, molecular and genetic studies were carried out to elucidate the regulatory role of COP1 in ABA signaling.
The cop1 mutants are hyposensitive to ABA-triggered stomatal closure under light and dark
conditions. COP1 interacts with and ubiquitinates the Arabidopsis clade A type 2C phosphatases (PP2Cs) ABI/HAB group and AHG3, thus triggering their degradation. Abscisic acid
enhances the COP1-mediated degradation of these PP2Cs. Mutations in ABI1 and AHG3
partly rescue the cop1 stomatal phenotype and the phosphorylation level of OST1, a crucial
SnRK2-type kinase in ABA signaling.
Our data indicate that COP1 is part of a novel signaling pathway promoting ABA-mediated
stomatal closure by regulating the stability of a subset of the Clade A PP2Cs. These findings
provide novel insights into the interplay between ABA and the light signaling component in
the modulation of stomatal movement.
Introduction
Stomata, consisting of guard cells, are highly specialized epidermal structures that regulate gas exchange and water transpiration
in terrestrial plants. Stomatal aperture is modulated through
changes in the intracellular turgor of guard cells to balance the
uptake of CO2 for photosynthesis and maintain water status in
response to environmental conditions such as light intensity,
water availability, CO2 concentration, etc. (Assmann, 1993;
Blatt, 2000; Schroeder et al., 2001; Shimazaki et al., 2007). The
stomatal response to these environmental conditions is often
mediated by the phytohormone abscisic acid (ABA), and the
underlying molecular mechanisms have been extensively studied
(Kim et al., 2010).
Abscisic acid promotes stomatal closure and inhibits light-induced stomatal opening. Perception of ABA in guard cells is
dependent on the pyrabactin resistance (PYR)/PYR1-like (PYL)/
regulatory components of ABA receptor (RCAR) protein family
*These authors contributed equally to this work.
of ABA receptors (Lee et al., 2013). The binding of ABA to PYR/
PYL/RCAR receptors recruits type 2C protein phosphatase
(PP2C) co-receptors, resulting in the release of PP2Cs from the
inhibitory PP2Cs-Open stomata 1 (OST1) complex (Ma et al.,
2009; Xie et al., 2012; Wang et al., 2018). The kinase activity of
OST1, an SNF1-related protein kinase 2 (SnRK2), is inhibited
when complexed with PP2Cs, but the recruitment of clade A
PP2Cs upon ABA binding with the ABA receptor de-represses
OST1 activity (Soon et al., 2012). The activated OST1 then
phosphorylates downstream targets, including the plasma membrane NADPH oxidase and Ca2+ channels, inducing the subsequent accumulation of second messengers such as reactive oxygen
species (ROS) and Ca2+ in the guard cells (Song et al., 2014).
These second messengers trigger signaling pathways leading to
the physiological responses to ABA (Kim et al., 2010; Kollist
et al., 2014; Zhang et al., 2014). In addition, OST1-mediated
phosphorylation activates the S-type slow anion channel-associated 1 (SLAC1) and the R-type quickly-activating anion channel
1 (QUAC1) but inactivates the K+ inward rectifying channel
(KAT1) in Arabidopsis. These phosphorylation events cause a
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massive outflow of NO3 and Cl anions from the guard cells,
while inhibiting K+ influx into them. As a result, the turgor pressure of the guard cells decreases and the stomata close (Lee et al.,
2009; Sato et al., 2009; Imes et al., 2013).
In contrast to ABA, light is one of the most important environmental signals that induces stomatal opening. Stomatal
opening during the day is mainly dependent on the coordination of red and blue light (Shimazaki et al., 2007). High-intensity red light induces stomatal opening by stimulating the
production of osmotic metabolites, thus maintaining the turgor of guard cells (Ogawa et al., 1978; Shimazaki et al., 2007).
However, the pathway used by red light receptors in guard
cells to regulate stomatal movement remains elusive. Compared to red light, which requires a prolonged time period for
the induction of stomatal opening, blue light induces stomatal
opening in a much faster way. Upon perception by the receptors, blue light quickly induces the phosphorylation of type 1
protein phosphatase (PP1), type 7 protein phosphatase (PP7)
and blue light signaling 1 (BLUS1), promoting H+-ATPase
activity on the cell membrane of guard cells (Takemiya et al.,
2006; Sun et al., 2012; Takemiya et al., 2013). The activated
H+-ATPase results in a massive outflow of H+, leading to
hyperpolarization of the guard cell membrane. The elevated
membrane potential activates the voltage-gated K+ channels,
and the resulting K+ influx increases the turgor of the guard
cells, which promotes stomatal opening (Shimazaki et al.,
2007). Although light and ABA regulate stomatal movement
by modulating the turgor pressure of guard cells, the mechanistic relationship between light and ABA signaling in stomatal
movement is not fully understood.
CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1)
is a master negative regulator of photomorphogenesis and plays
an important role in the transduction of red, far-red, blue and
ultraviolet (UV) light signals (Lau & Deng, 2012). COP1 is an
E3 ubiquitin ligase composed of a RING domain, a coiled helix
domain and a WD40 domain, and mediates the ubiquitination
and subsequent degradation of several regulatory factors in plant
photomorphogenesis (Torii et al., 1998). In dark conditions,
COP1 and Suppressor of phya-105 (SPA) form hetero-complexes in the nucleus, mediating the ubiquitination of transcription factors such as Elongated hypocotyl 5 (HY5), HY5
homologue (HYH), Long hypocotyl in far-red light 1 (HFR1),
and Long after far-red light 1 (LAF1) for degradation, thus
inhibiting photomorphogenesis and promoting skotomorphogenesis in plants (Osterlund et al., 2000; Holm et al., 2002; Seo
et al., 2003; Duek et al., 2004; Balcerowicz et al., 2017). In the
light, COP1 is exported from the nucleus to the cytosol, resulting
in the accumulation of these transcription factors and promoting
photomorphogenesis (Podolec & Ulm, 2018). In recent decades,
the functions of COP1 in light signaling have been extensively
studied, and accumulating evidence has shown that COP1 is also
involved in flowering, biological clock rhythm, virus defense,
plant hormone signaling, stomatal development and closing, and
other biological processes (Mao et al., 2005; Kang et al., 2009;
Wang et al., 2019). However, the role of COP1 in ABA signaling
remains unexplored.
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In this study, we demonstrate that COP1 is involved in ABAmediated stomatal closure. In cop1 mutants, impaired ABA-promoted stomatal closure is evident, and genetic analyses indicate
that COP1 controls stomatal movement upstream of the clade A
PP2Cs, which function as ABA signaling regulators. We show
that COP1 ubiquitinates ABI/HAB and AHG3 PP2Cs, triggering their degradation, and the COP1 ubiquitination of these
PP2Cs is enhanced by ABA. The COP1-mediated ubiquitination
and degradation of the PP2Cs, which are key players in ABA signaling, provides a novel pathway in the modulation of stomatal
closing.
Materials and Methods
Plant materials and growth conditions
All the genotypes used in this study were generated in the
Columbia (Col-0) ecotype background. The mutants used in this
study include cop1-4 (Ma et al., 2002), cop1-6 (Ma et al., 2002),
and GUS-COP1 (Osterlund & Deng, 1998). The T-DNA
insertion mutants abi1-3 (SALK_076309) and ahg3-1
(SALK_028132) were obtained from the Arabidopsis Biological
Resource Center (ABRC; http://abrc.osu.edu/). cop1-4abi1-3,
cop1-4ahg3-1, and cop1-4abi1-3ahg3-1 mutants were generated
by genetic crossing. The primers for identification of these
mutants are listed in Supporting Information Table S1. The
cop1-4 plants expressing 35S:ABI1-MYC or 35S:AHG3-MYC
were generated by crossing the 35S:ABI1-MYC or 35S:AHG3MYC transgenic plants with cop1-4 mutants. The seedlings were
grown in a 1 : 1 mixture of forest soil and vermiculite under
150 lmol photons m 2 s 1 illumination at 22°C with a 12 h :
12 h, light : dark photoperiod in a glasshouse.
Measurements of stomatal aperture
Measurements of stomatal aperture were performed as described
previously (Wang et al., 2020). Briefly, epidermal peels were
stripped from leaves of 4-wk-old plants grown in a glasshouse
under the conditions described in the previous paragraph, and
incubated in the resting buffer for 2.5 h under a light intensity of
150 lmol m 2 s 1 or in the dark with or without 1 lM ABA.
This experiment was repeated three times, and four to six independent plants were used in each experiment.
Bimolecular fluorescence complementation (BiFC) analysis
The coding sequences of the PP2Cs (ABI1, ABI2, HAB1, HAB2,
AHG3) with the N-terminal fragments of GFP (nGFP-COP1)
and COP1 with the C-terminal fragments of GFP (cGFP-COP1)
were cloned into the pCAMBIA1300 vector to create fusion
genes. The PP2C family member PP2C5, which is incapable of
interacting with COP1, was used as a negative control, and the
COP1-interacting protein HY5 was used as a positive control
(Kudla & Bock, 2016). The combinations of cGFP-COP1 with
nGFP-PP2Cs were co-transformed into Nicotiana benthamiana
leaves. H2B-mCherry was expressed in N. benthamiana leaves as
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a marker protein for nuclear localization (Guo et al., 2018).
Three days after the first injection, a second injection was carried
out with infiltration buffer containing 50 lM MG132. The
tobacco leaves were then cultured for 12 h, and the fluorescence
images were captured using a Zeiss LSM 710 scan confocal
microscope (Heidenheim, Germany). Primers used for the BiFC
assays are listed in Table S1.
Co-immunoprecipitation (Co-IP) assay in Arabidopsis
protoplasts
The coding sequences of COP1 with a CFP tag (CFP-COP1)
and PP2Cs (ABI1, ABI2, HAB1, HAB2, AHG3) with an MYC
tag (PP2Cs-MYC) were cloned into the pCAMBIA1300 vector
to create fusion genes, and these plasmids were co-transformed
into Arabidopsis protoplasts. The protoplasts were lysed by sonication in protein extraction buffer containing 50 mM Tris-HCl
(pH 7.4), 150 mM NaCl, 10 mM MgCl2, 0.2% (v/v) glycerol,
0.1% (v/v) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride
(PMSF) and 1 9 complete protease inhibitor (Roche), and then
centrifuged at 12 000 g for 15 min at 4°C. The supernatant was
incubated with 15 ll anti-GFP antibody-conjugated agarose
(Chromotek) for 3 h at 4°C. The Co-IP products were washed
briefly with extraction buffer three times at 4°C, and immune
blots with anti-MYC antibody and anti-GFP antibody were carried out. To test the effect of ABA on the interactions between
COP1 and PP2Cs, a final concentration of 0, 1, or 10 lM ABA
was added to the supernatant incubated with anti-GFP antibodyconjugated agarose. Primers used for Co-IP assays are listed in
Table S1.
Semi-in vivo protein degradation analysis
Semi-in vivo protein degradation analysis was performed as
described previously (Liu et al., 2010). Tobacco leaves were
injected with Agrobacterium carrying CFP-COP1, ABI1-MYC or
AHG3-MYC. The leaves without the Agrobacterium injection
(wild-type, WT) were also collected as controls. Proteins were
extracted from the leaves using the extraction buffer described in
the subsection ‘Co-immunoprecipitation (Co-IP) assay in Arabidopsis protoplasts’, above. A final concentration of 10 lM
ATP was added to the cell lysates to maintain the function of the
26S proteasome. The extract containing ABI1-MYC was mixed
with the extract containing CFP-COP1 or WT extract at a ratio
of 1 : 1, and the extract containing AHG3-MYC was also mixed
with the extract containing CFP-COP1 or WT extract at 1 : 1
ratio. These mixtures were either supplemented with 50 lM
MG132 or not supplemented. The mixture was incubated at
room temperature and sampled at 0, 2, 4 and 6 h.
For semi-in vivo degradation analysis of ABI1-MYC and
AHG3-MYC in WT and pyr1pyl1pyl2pyl4pyl5pyl8 (hexpyl ) backgrounds, 10 lg Pro35S:CFP-COP1 plasmid and 10 lg Pro35S:
ABI1-MYC plasmid, 10 lg Pro35S:CFP-COP1 plasmid and
10 lg Pro35S:AHG3-MYC plasmid were mixed and co-transformed into WT and hexpyl mutant protoplasts respectively.
After incubation for 16 h, total proteins were extracted using the
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extraction buffer and divided into three groups: one without
ABA treatment, one with 10 lM ABA and the other with 10 lM
ABA plus 50 lM MG132. The total proteins were incubated at
room temperature and sampled at 0, 4.5 and 9 h.
In vitro ubiquitination assays
In vitro ubiquitination assays were performed as described previously (Saijo et al., 2003; Liu et al., 2010; Xu et al., 2014), and
GST-ABI1 and GST-AHG3 proteins were acquired using these
methods. The primers for GST-ABI1 and GST-AHG3 are listed
in Table S1. CFP and CFP-COP1 samples were extracted from
N. benthamiana leaves in which they were transiently expressed.
CFP was used as a negative control. The cell lysates were then
mixed and immunoprecipitated with 15 ll anti-GFP antibodyconjugated agarose. The immunoprecipitated products were
washed briefly with extraction buffer three times at 4°C. The
ubiquitination reaction mixtures (60 ll) contained 30 ng of
UBE1 (E1; Boston Biochem, Cambridge, MA, USA), 30 ng of
UbcH5b (E2; Boston Biochem), 10 lg of HA-tagged ubiquitin
(HA-Ub; Boston Biochem), 200 ng of GST-ABI1 or GSTAHG3, and 3 ll immunoprecipitated product in a reaction buffer
containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, and
10 mM ATP. After 2 h of incubation at 30°C, the reactions were
immunoblotted with anti-GST antibody and anti-HA antibody.
In vivo ubiquitination analysis
In vivo ubiquitination assays were performed as described previously (Zhang et al., 2013). The coding sequences of ABI1 with
an MYC tag (ABI1-MYC) and AHG3 with an MYC tag (AHG3MYC) were cloned into the pCAMBIA1300 vector under the
control of the 35S promoter. Five-week-old N. benthamiana
leaves were used for the transient expression assay. Three days
after the first infiltration, a second infiltration was carried out
with infiltration buffer with or without 100 lM MG132 and
then cultured for 12 h before sample harvesting. Three grams of
leaf tissue from each sample was homogenized in a protein extraction buffer as described previously (Zhang et al., 2013). The
supernatant (with the equal volume and the equal amount of proteins) was incubated with 15 ll anti-MYC antibody-conjugated
agarose (Chromotek) for 3 h at 4°C. The immunoprecipitated
products were washed briefly with extraction buffer three times at
4°C, and then immunoblotted with anti-MYC antibody and
anti-Ub antibody. For the experiments investigating the effects of
ABA on the ubiquitination of ABI1 and AHG3, the first infiltration was followed by dark treatment for 3 d and then the second
infiltration with the buffer containing 100 lM MG132 with or
without 30 lM ABA. The samples were then cultured for 12 h
before harvest. ACTIN protein was used as a loading control.
Protein extraction and Western blot analysis
Transgenic seedlings with the WT background (35S:ABI1-MYC
or 35S:AHG3-MYC) and cop1-4 (35S:ABI1-MYC or 35S:AHG3MYC) were cultured under normal growth conditions for 5 d and
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subsequently transferred to media containing 100 lM CHX with
or without 10 lM ABA, or 100 lM CHX plus 50 lM MG132
with or without 10 lM ABA for 3 h. Total proteins were
extracted and quantified using the Bradford assay. The same
amount of proteins from each sample were separated using
sodium dodecyl sulphate-polyacrylamide gel electrophoresis
(SDS-PAGE). Western blotting was performed using anti-MYC
antibodies. Actin was used as the loading control.
Thermal imaging
Thermal imaging was performed as described previously (Dong
et al., 2018). Wild-type, cop1-4, abi1-3, ahg3-1, cop1-4abi1-3,
cop1-4ahg3-1 and cop1-4abi1-3ahg3-1 seedlings grown in normal
medium for 4 d were transplanted into soil with sufficient moisture. Seedlings were not watered and the soils were naturally
dried. After c. 20 d, thermal images were acquired within the
growth chamber using a ThermaCAMSC1000 infrared camera
(FLIR System, Danderyd, Sweden). Meanwhile, the sixth leaf of
each genotype was selected for far-infrared photography. Images
were analyzed using the image-analysis program IRWIN REPORTER
(v.5.31).
Phosphatase assay
The phosphatase activity was determined by using the Serine/
Threonine Phosphatase Assay System (Promega) according to
the manufacturer’s instructions. Briefly, 4-wk-old rosette leaves
of WT, cop1-4, cop1-4abi1-3ahg3-1, cop1-4abi1-3, cop1-4ahg31 and hexpyl were harvested and incubated in stomatal solution
with or without 10 lM ABA for 1 h under light. Total proteins were extracted with the buffer containing 100 mM Hepes
(pH 7.8), 5 mM EDTA, 5 mM EGTA, 10 mM sodium orthovanadate (Na3VO4), 10 mM sodium fluoride (NaF), 0.5%
NP-40, 5 mM okadaic acid, 50 ng ll 1 PPase-1 inhibitor-1,
and 1% protease inhibitor cocktail and filtered to remove inorganic phosphate (Pi) ions using Sephadex® G-25 resin. Total
proteins were quantified using the Bradford method. One-hundred micrograms of protein was used to perform the phosphatase assay.
Detection of phosphorylation level of OST1
To detect the phosphorylated OST1 protein in vivo, 4-wk-old
rosette leaves of WT, cop1-4, cop1-4abi1-3ahg3-1, abi1-3,
ahg3-1, hexpyl and ost1 were harvested and incubated in stomatal solution with or without 100 lM ABA for 1 h under light.
Proteins were extracted from the seedlings using the extraction
buffer described in the subsection ‘Co-immunoprecipitation
(Co-IP) assay in Arabidopsis protoplasts’, above. After proteins
were transferred to a PVDF membrane, total OST1 protein
was detected using anti-OST1 antibodies (Agrisera, Vannas,
Sweden) and served as loading control, while phosphorylated
OST1 was detected with an anti-pOST1 specific antibody
(kindly provided by Yang Zhao at the Shanghai Center for
Plant Stress Biology).
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Results
The cop1 mutants show defects in ABA regulated stomatal
movement
Previous studies demonstrated that COP1 constitutively inhibits
stomatal opening in both darkness and light, and that COP1 is
involved in ABA-mediated microtubule depolymerization and
stomatal closure (Mao et al., 2005; Khanna et al., 2014). In this
study, we also observed differences between the cop1 mutants and
WT in stomatal responses to ABA (Fig. 1a,b). Treatment of
WT, cop1-4 and cop1-6 mutants (McNellis et al., 1996) and the
cop1 functional complementation line GUS-COP1 (Osterlund &
Deng, 1998) with white light at an intensity of
150 µmol m 2 s 1 intensity for 2.5 h induced full stomatal opening in all genotypes (Fig. 1a). Nevertheless, both cop1 mutants
showed statistically significant increases in stomatal aperture
compared to WT plants (Fig. 1b). When plants were treated with
ABA (1 lM for 2 h), the cop1 mutant lines showed strongly
impaired ABA-induced stomatal closure compared to WT
(Fig. 1a,b), with width : length ratios of 0.573 0.05,
0.584 0.056 and 0.307 0.051 in cop1-4, cop1-6 and WT,
respectively (Fig. 1b). The complementation line GUS-COP1
phenocopied WT plants in all treatments (Fig. 1a,b). These
results indicate that the cop1 mutants are hyposensitive to ABAmediated stomatal responses under light. In addition, the ABAhyposensitivity of the cop1 mutant was also observed in root
growth and biomass measures at the seedling stage (Fig. S1).
To determine whether the ABA hyposensitivity of cop1
mutants is light-dependent, we measured stomatal responses to
ABA for all genotypes in the dark and observed a strong reduction in stomata aperture in WT and GUS-COP1 plants. By contrast, the stomatal response of cop1 mutants to ABA was again
significantly impaired (Fig. 1a,b). Together, our results indicate
that COP1 plays an important role in ABA-induced stomatal closure, which seems to be independent of COP1-mediated light
signaling.
COP1 interacts with the ABA signaling components ABI1,
ABI2, HAB1, HAB2 and AHG3
To study the nature of COP1 involvement in ABA-regulated
stomatal closure, yeast two-hybrid (Y2H) assays were used to test
the interaction between COP1 and known regulatory components of the ABA signaling pathway controlling stomatal movement (Table S2). Strong interactions were detected between
COP1 and several PP2Cs with critical roles in ABA signaling,
including ABA insensitive 1 (ABI1), ABA insensitive 2 (ABI2),
Hypersensitive to ABA1 (HAB1), Homology to ABI2 (HAB2)
and ABA-hypersensitive germination 3 (AHG3) (Fig. S2a). We
also verified that none of the nine PP2Cs examined here exhibited self-activation activity in yeast (Fig. S2b).
The COP1-PP2C interactions were also tested using BiFC
assays in N. benthamiana leaves. Green fluorescence signal was
visible in N. benthamiana leaves co-expressing cGFP-COP1 and
nGFP-PP2Cs, while no signal was detected in those coÓ 2020 The Authors
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WT
(a)
cop1-4
cop1-6 GUS-COP1
– ABA
Light
+ ABA
HAB2 and AHG3) fusion proteins in Arabidopsis protoplasts
and performed co-immunoprecipitation (Co-IP) analysis. The
results showed that CFP-COP1 co-precipitated with ABI1MYC, ABI2-MYC, HAB1-MYC, HAB2-MYC and AHG3MYC (Fig. 2b–f). Together, our results indicate that COP1
directly interacts with multiple clade A PP2Cs (ABI1, ABI2,
HAB1, HAB2 and AHG3) that play critical roles in ABA signaling.
COP1 ubiquitinates ABI1 and AHG3
Darkness
– ABA
+ ABA
Stomatal aperture (width/length)
(b)
0.90
0.75
*
*
WT
cop1-4
cop1-6
GUS-COP1
**
**
0.60
0.45
*
*
0.30
**
**
0.15
0.00
–ABA
+ABA
Light
–ABA
+ABA
Darkness
Fig. 1 Abscisic acid (ABA) induced stomatal responses of wild-type (WT),
cop1 mutants andGUS-COP1complementation plants in light and dark
conditions. (a) Representative images showing stomatal apertures of
Arabidopsis. Bar, 10 lm. (b) Quantification of stomatal apertures. Data are
mean values SD; n = c.150 stomata for three independent experiments.
*, P < 0.05; **, P < 0.01; Student’s t-test.
expressing cGFP-COP1 and nGFP-PP2C5 control (Fig. 2a). Coexpression of the nuclear fluorescence marker H2B-mCherry
revealed that the interactions of COP1 with ABI1, ABI2, HAB1
and HAB2 occurred in both cytoplasm and nucleus, whereas the
interaction between COP1 and AHG3 was restricted only to the
nucleus (Figs 2a, S3a). The punctuated fluorescence showing the
interactions between COP1 and these PP2Cs is similar to the
interaction of COP1 with HY5 (Fig. S3a), which is consistent
with the previously reported localization patterns of COP1 in
plant cells (Liu et al., 2008; Park et al., 2017; Swain et al., 2017).
Consistent with the Y2H results, AHG1, HAI1, HAI2 and HAI3
showed no interactions with COP1 in N. benthamiana leaves
(Fig. S3b). However, no direct interactions were found between
COP1 and ABA receptors using Y2H and BiFC (Fig. S4)
To further verify the interactions in vivo, we transiently
expressed CFP-COP1 and/or PP2Cs-MYC (ABI1, ABI2, HAB1,
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Since COP1 interacts with ABI1, ABI2, HAB1, HAB2 and
AHG3, we reasoned that these PP2Cs could be substrates of
COP1. We chose ABI1 and AHG3 for further study as they are
involved in the regulation of stomatal movement (Kuhn et al.,
2006; Hua et al., 2012) and show distinct interaction patterns
(Fig. 2a). We first tested whether COP1 can affect the stability of
ABI1 and AHG3 proteins using a semi-in vivo degradation assay.
As shown in Fig 3a, the presence of COP1 strongly enhanced the
degradation of ABI1-MYC and AHG3-MYC, while addition of
MG132, a commonly used inhibitor of proteasome-mediated
degradation, reduced the effect of COP1. These results suggest
that COP1 promotes the degradation of ABI1 and AHG3, probably through the ubiquitination pathway. We therefore performed in vitro ubiquitination analysis using GST-ABI1 and
GST-AHG3 fusion proteins purified from Escherichia coli as substrates. In the assays, HA-tagged ubiquitin was used as a co-substrate and CFP as a negative control. As shown in Fig. 3(b,c),
ABI1 and AHG3 were detected by both anti-GST and anti-HA
antibodies only when E1, E2, CFP-COP1 and HA-tagged ubiquitin were present, indicating that both proteins were ubiquitinated by E1, E2 and E3 (CFP-COP1) ligase. The size
distribution of GST-ABI1 and GST-AHG3 detected by both
anti-GST and anti-HA antibodies indicates polyubiquitination
of ABI1 and AHG3 by COP1. Polyubiquitinated COP1 was also
detected (Fig. 3b,c), which is consistent with a previous report
showing that the E3 ligase COP1 can be self-ubiquitinated in
such reactions (Seo et al., 2003).
To further verify the ubiquitination of ABI1 and AHG3 by
COP1, we co-expressed CFP-COP1 with ABI1-MYC or AHG3MYC in tobacco leaves and examined the ubiquitin status of
ABI1 and AHG3 in the presence or absence of MG132. After coexpression, total leaf proteins were immunoprecipitated using
anti-MYC antibodies, and the precipitated proteins were detected
by anti-MYC and anti-Ub antibodies. In the absence of MG132
treatment, ABI1 protein levels in the tobacco leaves co-expressing
CFP-COP1 and ABI1-MYC were lower than those in the
tobacco leaves expressing ABI1-MYC alone (Fig. 3d). Similarly,
co-expression of CFP-COP1 and AHG3-MYC produced lower
AHG3 levels than expression of AHG3-MYC alone (Fig. 3e).
These results suggest that COP1 mediates ABI1 and AHG3
degradation in plant cells. Interestingly, in the presence of
MG132, tobacco leaves co-expressing CFP-COP1 and either
ABI1-MYC or AHG3-MYC accumulated more polyubiquitinated ABI1-MYC or AHG3-MYC than leaves expressing ABI1MYC alone or AHG3-MYC alone (Fig. 3d,e), likely due to
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(b)
(c)
+
–
+
+
–
+
α-MYC
IP:
α-GFP
Merge
α-MYC
Fluorescence
IP:
α-GFP
Input
CFP-COP1
HABI1-MYC
kDa
Merge
nGFP-PP2C5
–
+
α-GFP
100
α-MYC
α-GFP
100
α-MYC
45
+
–
+
+
–
+
CFP-COP1
HABI2-MYC
kDa
α-GFP
100
70
α-MYC
α-GFP
100
70
α-MYC
100
70
100
70
+
–
+
+
–
+
α-GFP
α-MYC
α-GFP
α-MYC
(f)
CFP-COP1
AHG3-MYC
kDa
Input
Fluorescence
+
–
+
+
–
+
α-GFP
100
45
Merge
cGFP-COP1 + H2B-mCherry
nGFP-AHG3
+
+
(e)
IP:
α-GFP
cGFP-COP1 + H2B-mCherry
(d)
+
–
45
α-GFP
100
45
nGFP-HAB2
Input
α-GFP
100
45
nGFP-HAB1
CFP-COP1
ABI2-MYC
kDa
IP:
α-GFP
Input
Fluorescence
CFP-COP1
ABI1-MYC
kDa
Input
nGFP-ABI2
IP:
α-GFP
nGFP-ABI1
cGFP-COP1 + H2B-mCherry
(a)
100
45
α-MYC
α-GFP
α-MYC
Fig. 2 COP1 interacts with multiple PP2Cs in vivo. (a) Confocal images showing the bimolecular fluorescence complementation (BiFC) assay for the
interactions of COP1 with PP2Cs. The nGFP-PP2Cs and cGFP-COP1 were co-expressed in tobacco (Nicotiana benthamiana) leaves. nGFP-PP2C5 and
cGFP-COP1 were co-expressed in N. benthamiana as a negative control. H2B-mCherry was used as a nuclear localization marker. Green fluorescence
indicates interaction between the proteins. Red fluorescence shows the location of the nucleus. Bar, 50 lm. (b–f) Co-IP assays. A CFP-COP1 fusion protein
was co-expressed with the respective PP2C-MYC fusions in Arabidopsis protoplasts. Total protein extracts from transformed protoplasts were
immunoprecipitated using anti-GFP antibody-conjugated agarose and detected by immunoblot using anti-MYC antibody and anti-GFP antibody.
inhibition of degradation of ubiquitinated proteins caused by
MG132.
ABA promotes the degradation of ABI1 and AHG3 by
COP1
Recent studies have revealed that ABA promotes the degradation
of PP2Cs by the proteasome-mediated pathway (Kong et al.,
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2015; Wu et al., 2016). Since our results show that COP1 can
ubiquitinate ABI1 and AHG3, we tested the effect of ABA on the
COP1-mediated degradation of these PP2Cs. We first determined the impact of ABA on the interaction between COP1 and
either ABI1 or AHG3. Co-IP results showed that ABA treatment
increased the amount of ABI1-MYC and AHG3-MYC proteins
co-precipitating with COP1. In addition, the amount of ABI1MYC and AHG3-MYC proteins co-precipitated with COP1
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New Phytologist Ó 2020 New Phytologist Foundation
New
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Research 2041
MG132 + COP1
(a)
0
2
4
6
+ COP1
0
2
4
6
0
2
4
– MG132
+ MG132
+ MG132
ABI1-MYC – + + –
CFP-COP1 – – + +
kDa
180
– + + –
– – + +
– + + –
– – + +
(d)
– COP1
6
kDa
45
(h)
ABI1-MYC
α-MYC
43
α-ACTIN
100
45
AHG3-MYC
α-MYC
α-MYC
60
43
α-ACTIN
(b)
E1
E2
CFP-COP1
CFP
GST-ABI1
HA-Ubiquitin
+
–
–
–
+
+
+
+
–
–
+
+
+
+
+
–
+
+
+
+
–
+
+
+
+
+
–
+
–
+
+
+
+
–
–
+
(c)
E1 +
E2 –
CFP-COP1 –
CFP –
GST-AHG3 +
HA-Ubiquitin +
+
+
–
+
–
+
+
+
+
–
–
+
(e)
+ MG132
– + + –
– – + +
– + + –
– – + +
100
α-MYC
60
45
Ub(n)-CFP-COP1
130
α-HA
91
+ MG132
kDa
180
Ub(n)-GST-AHG3
Ub(n)-CFP-COP1
Ub(n)-GST-ABI1
70
180
– MG132
AHG3-MYC – + + –
CFP-COP1 – – + +
70
70
α-Ub
α-ACTIN
43
130
α-GST
91
130
α-GST
91
91
+
+
–
+
+
+
180
180
130
α-HA
+
+
+
–
+
+
kDa
kDa
180
+
+
–
–
+
+
45
α-Ub
43
α-ACTIN
70
Fig. 3 COP1 promotes ubiquitination and degradation of ABI1 and AHG3. (a) Semi-in vivo protein degradation assay showing increased degradation of
ABI1 and AHG3 in the presence of COP1. Protein extracts containing ABI1-MYC, AHG3-MYC or CFP-COP1 from transiently transformed tobacco
(Nicotiana benthamiana) leaves were used in this assay. Protein abundance was detected by immunoblotting. Actin was used as a loading control. (b, c) In
vitro ubiquitination assay showing COP1-mediated ubiquitination of ABI1 (b) and AHG3 (c). The recombinant GST-ABI1/GST-AHG3 fusion proteins were
expressed in Escherichia coli and purified. The protein extracts containing CFP-COP1 or CFP were purified from N. benthamiana leaves transiently
expressing each protein. E1 (UBE1), E2 (UBCh5b), and HA-tagged ubiquitin (Ub) were purchased from commercial companies. ABI1, AHG3 and COP1
ubiquitination was analyzed in immunoblots using anti-GST and anti-HA antibodies. (d, e) In vivo ubiquitination assays showing the ubiquitination of ABI1
(d) and AHG3 (e) by COP1. ABI1-MYC, AHG3-MYC and CFP-COP1 proteins were transiently expressed in N. benthamiana leaves and
immunoprecipitated using anti-MYC antibody-conjugated agarose. The gel blots were visualized using anti-MYC and anti-Ub antibodies. Actin was used
as a loading control.
increased with increasing ABA concentrations (Fig. 4a,b). Fluorescence intensities of the protoplasts co-expressing cLuc-COP1
and nLuc-ABI1, or cLuc-COP1 and nLuc-AHG3 were higher
than those of the protoplasts co-expressing nLuc-ABI1 and cLucEmpty, nLuc-AHG3 and cLuc-Empty, or nLuc-Empty and
cLuc-COP1 as indicated by split luciferase complementation
assay (Fig. S5). Moreover, fluorescence intensities of the protoplasts co-expressing cLuc-COP1 and nLuc-ABI1, or cLuc-COP1
and nLuc-AHG3 were further increased by treatment with
10 lM ABA (Fig. S5). These results suggest that ABA enhances
the association between COP1 and these two PP2Cs.
Ó 2020 The Authors
New Phytologist Ó 2020 New Phytologist Foundation
We then determined the effect of ABA on COP1-mediated
ubiquitination of PP2Cs in vivo. CFP-COP1 and either ABI1MYC or AHG3-MYC were co-expressed in tobacco leaves in the
presence or absence of ABA, and anti-MYC was used to
immunoprecipitate ABI1-MYC and AHG3-MYC. Western blotting of the immunoprecipitated proteins showed that ABA treatment increased the polyubiquitination levels of ABI1-MYC and
AHG3-MYC (Fig. 4c). These results indicate that ABA promotes
ubiquitination of ABI1 and AHG3 by COP1 in plant tissues.
To study whether ABA regulates ABI1 and AHG3 protein stability via COP1 in Arabidopsis, we generated transgenic plants
New Phytologist (2021) 229: 2035–2049
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New
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2042 Research
(a)
+
+
–
(c)
IP: α-MYC
ABI1-MYC –
AHG3-MYC +
ABA –
CFP-COP1 +
α-Ub
+
–
+
+
+
–
–
+
–
+
+
+
(b)
IP: α-GFP
+ + +
+ + +
– + ++
Input
+ +
+ +
+ ++
ABI1-MYC
CFP-COP1
ABA
kDa
α-MYC
45
α-GFP
100
–
+
–
+
+
+
–
(d)
CHX
+
–
+
+
+
–
–
+
–
+
+
+
Input
+ +
+ +
+ ++
AHG3-MYC
CFP-COP1
ABA
kDa
α-MYC
45
α-GFP
100
IP: α-GFP
+ + +
+ + +
– + ++
CHX + MG132
– ABA
+ ABA
– ABA
WT
WT
WT
+ ABA
WT
kDa
45
ABI1
α-MYC
43
α-ACTIN
60
45
α-MYC
AHG3
45
43
α-ACTIN
kDa
180
100
43
d
1.5
0.6
d
cd
bc
b
a
0.3
0.0
– – + + – – + +
– – – – + + + +
ABA
MG132
e
co
WT
p1
-4
-4
co
WT
p1
-4
co
WT
p1
WT
p1
-4
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
co
4
1co
p
p1
(f)
WT
c
c
1.2
0.9
WT
co
co
WT
p1
-4
-4
p1
co
WT
Normalized pixel intensity
(AHG3/ACTIN)
Normalized pixel intensity
(ABI1/ACTIN)
1.8
-4
α-ACTIN
(e)
e
d
de
c
bc
b
a
– – + + – – + + ABA
– – – – + + + + MG132
Fig. 4 Abscisic acid (ABA) promotes the degradation of PP2Cs by COP1 in vivo. (a, b) Immunoblot showing enhanced interaction between COP1 and
either ABI1 (a) or AHG3 (b) by ABA. 35S:CFP-COP1 and either 35S:ABI1-MYC or 35S:AHG3-MYC were co-expressed in Arabidopsis protoplasts in the
presence of different concentrations of ABA ( represents 0 lM, + represents 1 lM, ++ represents 10 lM). The total protein fraction was purified using
anti-GFP antibody-conjugated agarose and analyzed by immunoblotting using anti-MYC and anti-GFP antibodies. (c) In vivo ubiquitination assay showing
enhanced ubiquitination of ABI1 and AHG3 by COP1. Protein extracts from tobacco (Nicotiana benthamiana) leaves transiently co-expressing CFP-COP1
and either ABI1-MYC or AHG3-MYC, with or without 30 lM ABA treatment, were purified with anti-MYC antibody-conjugated agarose. The
immunoprecipitated proteins were analyzed by immunoblotting using anti-MYC and anti-Ub antibodies. Actin was used as a loading control. (d–f) Protein
stability assay showing increased stability of PP2Cs in cop1-4. Seedlings of 35S:ABI1-MYC and 35S:AHG3-MYC transgenic plants in the wild-type (WT)
and cop1-4 backgrounds were treated with 100 lM CHX ( ABA/+ABA) or 100 lM CHX plus 50 lM MG132 ( ABA/+ABA) and the total protein extracts
were analyzed by Western blotting with anti-MYC and anti-actin antibodies. Actin was used as a loading control. (e, f) Quantitative analysis of the signal
intensity shown in panel (d). Data are mean values SE of three replicates. Different letters indicate groupings of statistical differences at P < 0.05 (oneway ANOVA analysis).
constitutively expressing either ABI1-MYC or AHG3-MYC
fusion proteins (35S:ABI1-MYC or 35S:AHG3-MYC), and introduced the transgenes into the cop1-4 mutant background by
genetic crossing. In the absence of MG132, the cop1-4 mutant
accumulated more ABI1-MYC and AHG3-MYC than WT
(Fig. 4d–f). Abscisic acid-promoted degradation of ABI1-MYC
and AHG3-MYC in the cop1-4 background was less pronounced
than in WT plants, and ABA treatment resulted in a decrease of
ABI1-MYC protein by 38% in WT and by 16% in cop1-4. Similarly, AHG3-MYC protein level was decreased by 42% in WT
and by 23% in cop1-4 after ABA treatment (Fig. 4d–f). However,
when plants were treated with 50 lM MG132, which inhibits
proteasome-mediated protein degradation, there were no
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statistically significant differences in the ABI1-MYC and AHG3MYC levels between WT and cop1-4 backgrounds (Fig. 4e,f).
These results indicate that COP1 mediates the degradation of
ABI1 and AHG3 through the 26S proteasome pathway, and
ABA-enhanced ABI1 and AHG3 degradation is partly mediated
by COP1 in Arabidopsis.
COP1-mediated ubiquitination of ABI1 and AHG3 in ABA
signaling
Transient co-expression of COP1 with ABI1 or AHG3 in the
hexpyl ABA receptor mutant was used to determine whether the
ABA-promoted degradation of ABI1 and AHG3 by COP1 is
Ó 2020 The Authors
New Phytologist Ó 2020 New Phytologist Foundation
New
Phytologist
and the abi1-3 and ahg3-1 mutations reduced the phosphatase
activity of the cop1-4 mutant (Fig. 5c). These results further support the idea that COP1 mediates the degradation of ABI1 and
AHG3, thus affecting their phosphatase activity.
(a)
hexpyl
WT
MG132
– ABA + ABA + ABA
MG132
– ABA + ABA + ABA
0 4.5 9 4.5 9 4.5 9
0 4.5 9 4.5 9 4.5 9
(h)
ABI1
α-MYC
kDa
45
α-ACTIN
43
1.00 0.71 0.41 0.53 0.12 0.78 0.61 0.98 0.83 0.70 0.75 0.67 0.91 0.77
AHG3
α-MYC
α-ACTIN
45
43
1.00 0.84 0.51 0.73 0.31 0.88 0.81 1.01 0.83 0.67 0.79 0.54 0.91 0.79
(b)
Relative protein levels
(ABI1/ACTIN)
+ ABA
– ABA
1.2
MG132 + ABA
1.0
0.8
0.6
0.4
WT
hexpyl
0.2
0.0
1.2
Relative protein levels
(AHG3/ACTIN)
dependent on PYL ABA receptors . Wild-type and hexpyl mutant
protoplasts co-transformed with equal amounts of Pro35S:CFPCOP1, Pro35S:ABI1-MYC or Pro35S:CFP-COP1, Pro35S:
AHG3-MYC plasmids, respectively, were used to detect the abundance of ABI1-MYC and AHG3-MYC proteins (Fig. 5a). Quantitative measurements showed that the degradation rates of ABI1MYC and AHG3-MYC were markedly reduced in the hexpyl
mutant compared to WT with or without ABA treatment, and
the ABA-enhanced degradation of these PP2Cs in WT was also
alleviated in the hexpyl mutant (Fig. 5b). Treatment with MG132
significantly reduced the degradation rate of ABI1-MYC and
AHG3-MYC in both the WT and hexpyl mutant, suggesting that
the degradation is dependent on the 26S proteasome pathway.
Together, these results indicate that the ABA-promoted degradation of ABI1 and AHG3 via COP1 is partly dependent on PYLs.
We further examined the effect of COP1 on ABI1 and AHG3
enzyme activity. In the phosphatase activity assay, free phosphate
was hardly detected for GST, MBP or MBP-COP1, while a large
amount of free phosphate was released by the dephosphorylation
activity of GST-ABI1 or GST-AHG31, and the content of free
phosphate produced by ABI1 or AHG3 was significantly reduced
when MBP-COP1 was added (Fig. S6). This result indicates that
the presence of COP1 impairs the phosphatase activity of ABI1
and AHG3. To consolidate the role of COP1 in modulating
ABA signaling through PP2Cs and the downstream components,
we generated cop1-4abi1-3, cop1-4ahg3-1 and cop1-4abi1-3ahg31 mutants by genetic crossing (Fig. S7a–d) and measured the
phosphatase activity in WT, cop1-4, cop1-4abi1-3ahg3-1, cop14abi1-3, cop1-4ahg3-1, and hexpyl. The hexpyl mutant showed
the highest phosphatase activity among all genotypes, and the
cop1-4 mutant had significantly higher phosphatase activity than
WT with or without ABA treatment (Fig. 5c). Abscisic acid treatment reduced the phosphatase activity in all tested genotypes,
Research 2043
0 4.5 9
– ABA
0 4.5 9 (h)
MG132 + ABA
0 4.5 9
+ ABA
1.0
0.8
0.6
0.4
WT
hexpyl
0.2
0.0
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New Phytologist Ó 2020 New Phytologist Foundation
4.5 9
0
0
4.5 9
0
(c)
Phosphatase activity assay
(pmol phosphate min–1 μg–1 protein)
Fig. 5 COP1 mediates the ubiquitination of ABI1 and AHG3, which are
involved in the modulation of abscisic acid (ABA) signaling. (a) Semiin vivo degradation analysis of ABI1-MYC and AHG3-MYC in WT and
hexpyl backgrounds by co-expression with CFP-COP1. After incubation
for 16 h, the Arabidopsis protoplasts were treated with or without ABA, or
with ABA + MG132. An anti-MYC antibody was used to detect the ABI1MYC protein, and actin was used as a loading control. Values below the
images are the contents of ABI1-MYC or AHG3-MYC relative to wild-type
(WT) at each time point following the initiation of the experiment (0h). (b)
Quantitative analysis of the signal intensity of ABI1-MYC and AHG3MYC, respectively. (c) Phosphatase activity in WT, cop1-4, cop1-4abi13ahg3-1, cop1-4abi1-3, cop1-4ahg3-1 and hexpyl plants in the absence
or presence of 10 lM ABA. Total PP2C activity is represented by the
phosphatase activity assay, in which 5 mM okadaic acid was added to
inhibit the activity of PPP family Ser/Thr-specific phosphoprotein
phosphatases (e.g. PP1 and PP2A). Error bars represent SD (n = 6).
Different letters indicate groupings of statistical differences at P < 0.05
(one-way ANOVA analysis). (d) Detection of phosphorylated OST1 in
various genotype materials. Four-week-old rosette leaves of WT, cop1-4,
cop1-4abi1-3ahg3-1, abi1-3, ahg3-1, hexpyl and ost1 were harvested
and incubated in stomatal solution with or without 100 lM ABA for 1 h
under light. Total protein was extracted and total OST1 was used as a
loading control. Detection of OST1 was performed using an anti-OST1
antibody from Agrisera, and phosphorylated OST1 was detected using an
anti-pOST1 antibody.
15
f
12
e
e
9
6
9 (h)
cop1-4
cop1-4abi1-3
hexpyl
WT
cop1-4abi1-3ahg3-1
cop1-4ahg3-1
18
4.5
c
c
cd
d
cd
b
a
b bc
3
0
– ABA
+ABA
(d)
kDa
45
α-pOST1
45
α-OST1
0.00 0.00 0.00 0.00 0.00 0.00 1.00 0.54 0.85 1.21 1.16 0.21 0.00
– ABA
+ ABA
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We subsequently detected the phosphorylation levels of OST1
in the leaves of WT, cop1-4, cop1-4abi1-3ahg3-1, abi1-3, ahg3-1,
hexpyl and ost1 with or without ABA treatment. Two specific
antibodies have been used, and the anti-OST1 is an antibody
specific to OST1 protein, while the anti-pOST1 is an antibody
specific to phosphorylated OST1 protein. We observed that
OST1 protein was present in all genotypes except ost1, while
phosphorylated OST1 could only be detected after ABA treatment (Fig. 5d). Quantitative analysis showed that the level of
phosphorylated OST1 was lowest in hexpyl among all genotypes
and significantly reduced in cop1-4 when compared with WT,
while abi1-3 and ahg3-1 mutants showed higher levels of phosphorylated OST1 than WT (Fig. 5d). Although the level of phosphorylated OST1 in cop1-4abi1-3ahg3-1 was lower than that in
WT, the cop1-4abi1-3ahg3-1 mutant had higher phosphorylated
OST1 than the cop1-4 single mutant, indicating that the abi13ahg3-1 mutations could partially restore the phosphorylation
level of OST1 in cop1-4. Together, these results indicate that
COP1 modulates the PP2C activity and consequently the kinase
activity of OST1 to control stomatal movement.
The abi1-3 and ahg3-1 mutations suppress the hyposensitivity of cop1-4 mutants to ABA in stomatal movement
We tested whether loss-of-function mutations in ABI1 and AHG3
could rescue the stomatal response phenotype of the cop1 mutant
to ABA under light conditions. The reduced ABA sensitivity shown
by the cop1-4 mutant was restored to the WT level in the cop14abi1-3 and cop1-4ahg3-1 double mutants (Fig. 6a,b), indicating
that abi1-3 and ahg3 suppress the cop1-4 hyposensitivity to ABAmediated stomatal closing. Since the cop1 mutants are hyposensitive to ABA-mediated stomatal responses in both light and dark
conditions, we also examined the stomatal responses of these genotypes to ABA under dark conditions. The stomatal opening of
cop1-4 was still larger than that of other genotypes, and the stomatal opening of cop1-4abi1-3, cop1-4ahg3-1 and cop1-4abi1-3ahg31 were larger than that of the WT but significantly smaller than
that of cop1-4 under ABA treatment in darkness (Fig. S8). This
genetic evidence further supports the idea that ABI1 and AHG3
are downstream targets of COP1 and that the reduced sensitivity of
cop1 to ABA-mediated stomatal closing is due to decreased degradation of these PP2Cs.
Water loss was measured in detached leaves of soil grown WT,
cop1-4, abi1-3, cop1-4abi1-3, ahg3-1, cop1-4ahg3-1 and cop14abi1-3ahg3-1 mutants. As expected, cop1-4 mutants showed
higher water loss than WT (Fig. 6c). The increased water loss in
cop1-4 was only partially recovered in cop1-4abi1-3 and cop14abi1-3ahg3-1 mutants (Fig. 6c, measurement points starting 3 h
after detachment), indicating the involvement of additional
PP2Cs in the stomatal response. Stomatal density measurements
showed that the cop1-4 single mutant clearly had higher stomatal
density than WT, abi1-3 and ahg3, while the cop1-4, cop1-4abi13, cop1-4ahg3-1 and cop1-4abi1-3ahg3-1 mutants exhibited similar stomatal densities (Fig. S9). This result suggests that both
larger aperture and higher density of stomata in cop1-4 may contribute to higher water loss compared with WT, while the
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differences in water loss among the cop1-4, cop1-4abi1-3, cop14ahg3-1 and cop1-4abi1-3ahg3-1 mutants result merely from the
differences in stomatal aperture but not stomatal density.
We also measured the leaf temperatures of these genotypes
under drought conditions. The cop1-4 mutants showed statistically significant lower temperatures than WT, while the cop14abi1-3, cop1-4ahg3-1 and cop1-4abi1-3ahg3-1 mutants partially
rescued the decreases observed in cop1-4 temperatures (Fig. 6d–
g). In order to eliminate the influence of plant size on the temperature experiments, we selected the sixth rosette leaf, which is not
shaded by others, to observe the leaf temperature, and the leaf
temperature results with detached leaves resembled the whole
plant measurements (Fig. S10). Together, our results strongly
indicate that ABI1 and AHG3 are the direct downstream targets
of COP1 in the ABA-mediated control of stomatal movement.
Discussion
The COP1 protein is highly conserved in plants and mammals,
and its role in plant photomorphogenesis has been extensively studied (Wang et al., 1999; Lau & Deng, 2012). COP1 has also been
implicated in stomatal movement, with the cop1 mutant exhibiting
insensitivity of stomatal closing in response to darkness (Khanna
et al., 2014). Our findings also prove that ABA-induced stomatal
closure in cop1 mutants is greatly reduced compared to WT plants,
suggesting an important role for COP1 in ABA signaling. However, the molecular basis for the role of COP1 in the ABA control
of stomatal movement is still unknown. In this study, we show that
COP1 interacts with several clade A PP2Cs, resulting in their ubiquitination and degradation, which is enhanced by ABA, to potentiate the ABA-triggered stomatal closure.
This work provides several lines of evidence supporting a key
role for COP1 in the ABA-triggered stomatal closure. First, ABAinduced stomatal closure in cop1 mutants is severely reduced compared to wild-type plants. The ABA hyposensitivity in cop1-4 can
be partially recovered in cop1-4abi1-3, cop1-4ahg3-1 and cop14abi1-3ahg3-1 mutants, implying that ABI1 and AHG3 are downstream targets of COP1 in regulating stomatal movement. Second,
our in vitro and in vivo data show the interaction between COP1
and several clade A PP2Cs, including ABI1 and AHG3. Third, we
show that COP1 can ubiquitinate ABI1 and AHG3, promoting
their degradation. Importantly, ABA enhances the association of
COP1 with either ABI1 or AHG3, and promotes the ubiquitination and degradation of both PP2Cs. The ABA-induced reduction
of these PP2Cs by COP1 promotes stomatal closure in response to
stress conditions. In accordance, cop1 mutants do not efficiently
reduce PP2C levels in response to ABA and, as a consequence, fail
to accumulate high levels of phosphorylated OST1, thus inhibiting
stomatal closure.
The finding that COP1 acts as an upstream positive regulator
in ABA signaling raises the question of how the COP1-clade A
PP2C regulatory module fits into the ABA signaling pathway.
We propose a model in which COP1 recruits and degrades a subset of the clade A PP2C co-receptors in the ABA-PYR/PYL/
RCAR-PP2C-SnRK2 core signaling pathway (Fig. 7) promoting
stomatal closure. Our results showing that cop1 mutants show
Ó 2020 The Authors
New Phytologist Ó 2020 New Phytologist Foundation
New
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Research 2045
(a)
cop1-4
WT
cop1-4
abi1-3
abi1-3
cop1-4 cop1-4abi1-3
ahg3-1
ahg3-1
ahg3-1
– ABA
+ ABA
(c)
– ABA
0.4
+ ABA
a
0.3
b
0.2
b
b
b
b
**
** **
**
**
**
60
WT
cop1-4
abi1-3
cop1-4abi1-3
ahg3-1
cop1-4ahg3-1
cop1-4abi1-3
ahg3-1
40
20
0
0.0
-3
abi1-3
cop1-4
cop1-4
abi1-3
WT
ahg3-1
cop1-4
cop1-4
ahg3-1
-3
i1
ab
-4
p1 1
co g3- 1
ah g3
h
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(b)
cop1-4abi1-3
ahg3-1
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abi1-3
cop1-4
cop1-4
abi1-3
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cop1-4
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cop1-4abi1-3 20.6
ahg3-1
cop1-4abi1-3
ahg3-1
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cop1-4abi1-3
ahg3-1
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(g)
(f)
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23.0
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a
b
Leaf temperature (°C)
Leaf temperature (°C)
24.0
22.5
22.0
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WT
-1
g3
ah
-3
i1
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-4
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-3
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-4
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-4
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-3
i1
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WT
b
21.5
20.5
22.0
c
c
Fig. 6 The Arabidopsis abi1-3 and ahg3-1 mutations restore cop1-4 phenotypes. (a) Representative images showing the stomatal apertures of wild-type
(WT), cop1-4, abi1-3, ahg3-1, cop1-4abi1-3, cop1-4ahg3-1 and cop1-4abi1-3ahg3-1 in response to 1 lM ABA. Bar, 10 lm. (b) Quantitative analysis of
the stomatal apertures of the genotypes shown in (a). Data are mean values SD of three independent experiments. n = c.150 stomata. Different letters
indicate groupings of statistical differences at P < 0.05 (one-way ANOVA analysis). (c) Water loss of detached leaves of WT, cop1-4, abi1-3, ahg3-1,
cop1-4abi1-3, cop1-4ahg3-1 and cop1-4abi1-3ahg3-1. Three independent experiments were performed. The data are mean values SE of three
replicates. **, P < 0.01; Student’s t-test. (d, e) Representative false-color infrared images of WT, cop1-4, abi1-3, ahg3-1, cop1-4abi1-3, cop1-4ahg3-1,
and cop1-4abi1-3ahg3-1 plants withholding water for 20 d. Bars, 1 cm. (f, g) Quantitative analysis of leaf temperature of the genotypes shown in (c, d).
The leaf temperatures were calculated using IRWIN REPORTER v.5.31 software. Data are mean values SD (n = 30 plants for each condition; data are
from c. 4000 measurements of square pixels from multiple leaves of each plant). Different letters indicate groupings of statistical differences at P < 0.05
(one-way ANOVA analysis).
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ABA
+
+
COP1 PP2C
ABA
PP2C PYR/PYL/RCAR
PP2C
OST1
OST1
Stomatal closing
Fig. 7 A simplified model for the role of COP1 in abscisic acid (ABA)regulated stomatal closure in Arabidopsis. The clade A PP2Cs act as
negative regulators of stomatal closure mainly by inhibiting the activity of
OST1. Recruitment of the clade A PP2Cs by the ABA-PYR/PYL/RCAR
receptor pathway leads to de-repression of OST1 and stomatal closure
(right-hand pathway in the model schematic depicted in the figure).
COP1-mediated ubiquitination and subsequent degradation of the clade A
PP2Cs increases free OST1, leading to stomatal closure (left-hand
pathway). Abscisic acid can potentiate the effect of the COP1-PP2Cs
regulatory module. The ABA-promoted degradation of ABI1 and AHG3 by
COP1 is partly dependent on PYLs.
reduced, but not completely abolished, sensitivity to ABA-triggered stomatal closure support this model. The existence of several ABA-mediated signaling mechanisms provides redundancy,
and amplifies the speed and magnitude of the stress response.
The evolution of multiple mechanisms to regulate ABA co-receptors also reflects the importance and the complex dynamics controlling the regulation of stomatal movement.
COP1 is likely to play a role in the dynamic modulation
and homeostasis of clade A PP2C co-receptors in the cell, even
in the absence of stress, as it does not require the presence of
ABA for its interaction with ABI1 and AHG3 and their subsequent ubiquitination. Nevertheless, ABA greatly increases the
association of COP1 with these two substrates, and enhances
the ubiquitination of ABI1 and AHG3 by COP1, linking it to
the stress response. Indeed, previous studies have shown that
the expression of COP1 is induced by multiple stresses, including drought, salt and cold (Moazzam-Jazi et al., 2018). The
increased COP1 levels in response to stress could incite
increased COP1-mediated degradation of some of the clade A
PP2Cs promoting stomatal closure. Dynamic changes in PP2C
abundance could be important for adaptation of plants to
changing environmental conditions by timely and efficiently
modulating stomatal behavior.
Although we have proven the role of COP1 in the control of
ABI1 and AHG3 cellular levels, our protein interaction studies
show strong interaction with other PP2C co-receptors such as
ABI2, HAB1 and HAB2, which suggests that COP1 might also
be involved in their regulation. Indeed, the fact that the cop14abi1-3, cop1-4ahg3-1 and cop1-4abi1-3ahg3-1 mutants can only
partially rescue the cop1-4 phenotype indicates the involvement
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of additional PP2Cs in this process, suggesting the involvement
of other PP2Cs in regulating COP1-related stomatal movement.
According to sequence alignment and phylogenetic analysis,
clade A PP2Cs are divided into two groups, with ABI1, ABI2,
HAB1 and HAB2 in one and AHG1, AHG3, HAI1, HAI2 and
HAI3 in the other (Schweighofer et al., 2004; Antoni et al.,
2012). The interaction of COP1 with only five clade A PP2Cs
suggests that the interaction is selective. Similar selectivity has
been reported for several other clade A PP2C-interacting proteins. For example, the uncharacterized protein EAR1 (Enhancer
of ABA co-receptor 1) negatively regulates ABA signaling by
interacting with six PP2C members, but not HAI (Wang et al.,
2018). The E3 ligase PIR1.2 (RING finger protein1.2) interacts
strongly with PP2CA and AHG1, and weakly with HAI1 and
HAI3, but not with ABI/HAB PP2Cs (Baek et al., 2019). The
E3 ligases RGLG (RING DOMAIN LIGASE) 1 and 5 modulate
ABA signaling by controlling the turnover of AHG3, ABI2, and
HAB2 (Wu et al., 2016). In addition, DOG1 (Delay of germination1), RH8 (RNA helicase-like8), BPM (BTB/POZ AND
MATH DOMAIN protein) 3 and 5 all display selective interactions with members of the clade A PP2Cs (Nee et al., 2017; Baek
et al., 2018; Julian et al., 2019).
On the other hand, functional divergence may explain the
interaction of COP1 with AHG3 but not AHG1. Although both
AHG1 and AHG3 function in ABA signaling during seed development and germination, AHG3 also plays a role in stomatal
movement in response to ABA, while AHG1 is only expressed in
seeds, and mutations in AHG1 show no phenotypes in adult
plants (Nishimura et al., 2007; Antoni et al., 2012). Similarly,
the HAI group PP2Cs are functionally distinct from other members. HAI PP2C genes are strongly induced by stress or ABA, and
HAI PP2Cs participate in drought and osmotic stress response
mainly by negatively regulating the accumulation of osmolytes
such as proline and betaine in an ABA-independent manner. In
addition, the hai double and triple mutants show ABA-insensitive
seed germination, which is different from other clade A PP2C
members (Bhaskara et al., 2012). The distinct functions of HAI
from other clade A PP2Cs could explain the selective interaction
of COP1 with some of the members but not HAIs. Nevertheless,
detailed interaction analysis, including identifying the interacting
motifs in COP1 and the clade A PP2Cs, will be needed to further
understand the selective interaction of COP1 with the clade A
PP2Cs.
Furthermore, our data also suggest that COP1 might not be
the only E3 ligase involved in the degradation of clade A PP2Cs,
since the ABI1 and AHG3 levels still decreased in the cop1-4
mutant after ABA treatment. Our results are consistent with previous reports indicating that other E3 ligases, such as PUB12/13,
RGLG1/5, and BPM3/5, are also involved in the regulation of
the stability of PP2Cs (Kong et al., 2015; Wu et al., 2016; Julian
et al., 2019).
Further work is needed to determine which other ABA-mediated responses use the ABA-induced COP1 ubiquitination and
degradation of PP2C co-receptors as part of the signaling mechanism. COP1 has been implicated in the modulation of cytoskeletal processes by degradation of microtubule proteins and
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activation of the S-type anion channels. It will be interesting to
investigate whether the COP1-dependent degradation of ABI1
and AHG3, and possibly a whole subset of PP2C co-receptors,
may contribute to this process.
Our results indicate the involvement of COP1 in ABA-induced stomatal closure in both light and darkness, since stomatal
closure in cop1 mutants is still hyposensitive to ABA in dark conditions (Fig. 1). COP1 is a critical light signaling component,
and it accumulates in the nucleus in darkness, while visible light
promotes the nuclear export of COP1 (von Arnim et al., 1997;
Lau & Deng, 2012; Hoecker, 2017). We show that COP1 interacts with ABI1, ABI2, HAB1 and HAB2 in both the cytoplasm
and nucleus, providing a possible mechanism to explain the
involvement of COP1 in stomatal movement. Overall, our data
provide a mechanistic explanation positioning COP1 as a central
integrator of the crosstalk between ABA and light signaling in the
regulation of stomatal movement.
Acknowledgements
We thank Professor Qi Xie (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) for valuable discussion, Professor Xiaohong Zhu (Henan University) and Yang
Zhao (Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences) for providing the pyr1pyl1pyl2pyl4pyl5pyl8
(hexpyl ) seed and anti-pOST1 specific antibody. This work was
supported by National Natural Science Foundation of China
(31970198) and Key Project of Natural Science Foundation of
China (U1604233).
Author contributions
C-PS conceived and directed the project. C-PS, QC and LB
designed all experiments. QC performed all experiments with the
help of WW, QZ and KL. WW helped prepare the Arabidopsis
mutant materials used in this study. C-PS, LB, QC, and HS performed data analysis. HS, QC, LB, C-PS, JRB and H-QY wrote
the manuscript with the assistance and approval of all authors.
QC, LB and WW contributed equally to this work.
ORCID
Ling Bai https://orcid.org/0000-0001-9105-0377
Jose Ramon Botella https://orcid.org/0000-0002-4446-3432
Qingbin Chen https://orcid.org/0000-0003-2900-5852
Huazhong Shi https://orcid.org/0000-0003-3817-9774
Chun-Peng Song https://orcid.org/0000-0001-8774-4309
Wenjing Wang https://orcid.org/0000-0002-4513-2280
Hong-Quan Yang https://orcid.org/0000-0001-6215-2665
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Supporting Information
Additional Supporting Information may be found online in the
Supporting Information section at the end of the article.
Fig. S1 The cop1-4 mutant is hyposensitive to ABA in root
growth.
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Fig. S2 COP1 interacts with multiple PP2Cs in yeast.
Fig. S9 Stomatal density in the leaves of different genotypes.
Fig. S3 Bimolecular fluorescence complementation (BiFC) assays
of interactions between COP1 and the indicated PP2Cs in
Nicotiana benthamiana leaves.
Fig. S10 The leaf temperature of detached leaves.
Fig. S4 Interaction analysis of COP1 and PYLs.
Table S1 Primer sequences used in the manuscript.
Fig. S5 Split-luciferase complementation assay of interactions
between COP1 and ABI1/AHG3 in Arabidopsis protoplasts.
Table S2 Y2H screening results for COP1 and the known regulatory components of the ABA signaling pathway.
Fig. S6 COP1 affects the phosphatase activity of ABI1 and
AHG3 in vitro.
Please note: Wiley Blackwell are not responsible for the content
or functionality of any Supporting Information supplied by the
authors. Any queries (other than missing material) should be
directed to the New Phytologist Central Office.
Fig. S7 Identification of the double mutants and the triple
mutant by polymerase chain reaction (PCR) and quantitative real
time polymerase chain reaction (qRT-PCR).
Methods S1 Supplementary Materials and Methods.
Fig. S8 Stomatal opening of different genotypes under dark conditions.
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