Arabidopsis PLANT U-BOX44 down-regulates osmotic stress signaling by mediating Ca2+-DEPENDENT PROTEIN KINASE4 degradation

Abstract

Calcium (Ca2+)-dependent protein kinases (CPKs) are essential regulators of plant responses to diverse environmental stressors, including osmotic stress. CPKs are activated by an increase in intracellular Ca2+ levels triggered by osmotic stress. However, how the levels of active CPK protein are dynamically and precisely regulated has yet to be determined. Here, we demonstrate that NaCl/mannitol-induced osmotic stress promoted the accumulation of CPK4 protein by disrupting its 26S proteasome-mediated CPK4 degradation in Arabidopsis (Arabidopsis thaliana). We isolated PLANT U-BOX44 (PUB44), a U-box type E3 ubiquitin ligase that ubiquitinates CPK4 and triggers its degradation. A calcium-free or kinase-inactive CPK4 variant was preferentially degraded compared to the Ca2+-bound active form of CPK4. Furthermore, PUB44 exhibited a CPK4-dependent negative role in the response of plants to osmotic stress. Osmotic stress induced the accumulation of CPK4 protein by inhibiting PUB44-mediated CPK4 degradation. The present findings reveal a mechanism for regulating CPK protein levels and establish the relevance of PUB44-dependent CPK4 regulation in modulating plant osmotic stress responses, providing insights into osmotic stress signal transduction mechanisms.

Full text

Arabidopsis PLANT U-BOX44 down-regulates osmotic
stress signaling by mediating Ca
2+
-DEPENDENT
PROTEIN KINASE4 degradation
Wei Fan ,
1
Xiliang Liao ,
1
Yanqiu Tan ,
1
Xiruo Wang ,
1
Julian I. Schroeder
2
and Zixing Li
1,2,3,
*
1Joint Center for Single Cell Biology, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, China
2 Department of Cell and Developmental Biology, School of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA
3 Shanghai Collaborative Innovation Center of Agri-Seeds, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan
Road, Shanghai 200240, China
*Author for correspondence: zixing-li@sjtu.edu.cn
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the
Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is: Zixing Li (zixing-li@sjtu.edu.cn).
Abstract
Calcium (Ca
2+
)-dependent protein kinases (CPKs) are essential regulators of plant responses to diverse environmental stres-
sors, including osmotic stress. CPKs are activated by an increase in intracellular Ca
2+
levels triggered by osmotic stress. However,
how the levels of active CPK protein are dynamically and precisely regulated has yet to be determined. Here, we demonstrate
that NaCl/mannitol-induced osmotic stress promoted the accumulation of CPK4 protein by disrupting its 26S proteasome-
mediated CPK4 degradation in Arabidopsis (Arabidopsis thaliana). We isolated PLANT U-BOX44 (PUB44), a U-box type E3
ubiquitin ligase that ubiquitinates CPK4 and triggers its degradation. A calcium-free or kinase-inactive CPK4 variant was pref-
erentially degraded compared to the Ca
2+
-bound active form of CPK4. Furthermore, PUB44 exhibited a CPK4-dependent nega-
tive role in the response of plants to osmotic stress. Osmotic stress induced the accumulation of CPK4 protein by inhibiting
PUB44-mediated CPK4 degradation. The present findings reveal a mechanism for regulating CPK protein levels and establish
the relevance of PUB44-dependent CPK4 regulation in modulating plant osmotic stress responses, providing insights into os-
motic stress signal transduction mechanisms.
Received April 25, 2022. Accepted June 19, 2023. Advance access publication June 20, 2023
© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
Research Article
Introduction
Hyperosmotic stress caused by soil water deficit is one of the
most serious threats to plant survival. Upon exposure to hy-
perosmotic environments, plants trigger sophisticated signal-
ing cascades to adjust intracellular osmotic potential, reduce
the loss of water by transpiration, and enhance the capability
to explore water resources (Zhu 2016; Robbins and Dinneny
2018; Dinneny 2019; Zhang et al. 2022). Our understanding
of molecular osmotic stress perception and early signal trans-
duction mechanisms remains limited. Osmotic stress-induced
rapid elevation in cytosolic calcium (Ca
2+
) was discovered and
is well studied (Knight et al. 1997; Kudla et al. 2010, 2018;
Yuan et al. 2014; Stephan et al. 2016; Edel et al. 2017; Jiang
et al. 2019). Calcium-dependent protein kinases (CPKs) that
contain both a calcium-binding domain and a kinase domain
function as primary calcium signal decoders regulate plant
physiological responses to a wide range of abiotic/biotic stres-
ses (Harper et al. 2004; Franz et al. 2011; Boudsocq and Sheen
2013; Schulz et al. 2013; Brandt et al. 2015; Yip Delormel and
Boudsocq 2019; Schulze et al. 2021).
CPKs possess a conserved domain architecture that is com-
posed of a canonical Ser/Thr protein kinase domain and a
calmodulin-like Ca
2+
-binding domain usually containing 4
EF-hand (Calcium-binding Sites) motifs (CaM-like domain),
linked together by an autoinhibitory junction (AIJ) and flanked
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by variable amino and carboxyl termini (Harper et al. 1991,
1994; Harmon et al. 1994). As the name implies, most CPKs
require Ca
2+
for their activation (Boudsocq et al. 2012).
When Ca
2+
binds to all EF-hands in the CaM-like domain,
the calcium-dependent protein kinase (CPK) protein under-
goes a conformational change, thus releasing the kinase active
site from the repression of an autoinhibitory junction (Harmon
et al. 1994; Harper et al. 1994; Boudsocq et al. 2012; Ingram et al.
2015; Bender et al. 2018; Yip Delormel and Boudsocq 2019).
CPKs vary in terms of their sensitivity to Ca
2+
, presumably
allowing proteins to perceive distinct stimuli by differences in
Ca
2+
-binding affinity. For example, Arabidopsis (Arabidopsis
thaliana) CPK4 exhibits half maximal kinase activity in the pres-
ence of approximately 3 μM free Ca
2+
, while CPK5 only requires
about 100 nM (Guerra et al. 2020). In addition, lipid and 14-3-3
protein binding can potentiate calcium activation (Camoni
et al. 1998; Dixit and Jayabaskaran 2012; van Kleeff et al.
2018). Site-specific phosphorylation occurs in response to dis-
tinct stimuli that control CPK activities (Witte et al. 2010;
Bender et al. 2017; Ito et al. 2017; Bredow et al. 2021).
Activated CPKs become inactive under resting conditions
when cytosolic Ca
2+
concentration decreases (Harper et al.
1991; Boudsocq et al. 2012; Bender et al. 2018; Yip Delormel
and Boudsocq 2019). Similarly, the expression of stress-induced
CPKs reverts to basic levels as stress is relieved (Yip Delormel
and Boudsocq 2019). Furthermore, spliced variants and changes
in subcellular localization have been reported for several CPKs,
thus creating another layer of regulation for CPK activities
(Almadanim et al. 2018; Yip Delormel and Boudsocq 2019;
Medina-Puche et al. 2020). However, little is known about
CPK protein turnover in response to stress stimuli.
Ubiquitination is an important post-translational modifica-
tion that regulates the stability of protein substrates. The ubi-
quitination reaction involves 3 sequential steps catalyzed by a
ubiquitin-activating enzyme (E1), ubiquitin-conjugating en-
zyme (E2), and ubiquitin ligase (E3). The Arabidopsis genome
encodes more than 1,000 E3 ubiquitin ligases; these represent
key factors for determining the substrate specificity of the 26S
proteasome system. Recently, plant U-box (PUB) type E3 li-
gases have been demonstrated to be involved in drought/
salt stress and abscisic acid (ABA) signaling pathways.
Arabidopsis PUB22/23 and PUB11 negatively control a
drought signaling pathway by the ubiquitination of
REGULATORY PARTICLE NON-ATPASE 12A (RPN12a) and
LEUCINE RICH REPEAT PROTEIN 1 (LRR1)/KINASE 7
(KIN7), respectively (Cho et al. 2008; Chen et al. 2021).
PUB30 negatively regulates salt tolerance by facilitating the
degradation of BRI1 KINASE INHIBITOR 1 (BKI1) (Zhang
et al. 2017). The homologous PUB12/13 E3 ligases target
ABA INSENSITIVE 1 (ABI1) for degradation in the ABA signal-
ing pathway (Kong et al. 2015). PUB18/19 are involved in the
negative regulation of ABA-mediated stomatal movements
by facilitating the ubiquitination and protein turnover of
EXOCYST SUBUNIT EXO70 FAMILY PROTEIN B1 (Exo70B1)
(Seo et al. 2016). These extensive studies indicate that
drought could be a driver of PUB diversification during plant
terrestrialization (Trenner et al. 2022). PUB44, also referred to
as SAUL1 regulates senescence, cell death, and pathogen de-
fense (Tong et al. 2017). An autoimmunity-induced leaf ne-
crosis/seedling lethality phenotype in the saul1 mutant is
dependent on downstream PHYTOALEXIN DEFICIENT 4
(PAD4) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1)
and can be rescued by high temperature and high light con-
ditions (Vogelmann et al. 2012; Disch et al. 2016).
Interestingly, several molecular and genetic studies have im-
plicated the existence of crosstalk between osmotic stress
and pathogen defense responses (Kim et al. 2011; Feng
et al. 2018; Guo et al. 2018; Chen et al. 2020; Thor et al. 2020).
CPK4 is a positive regulator of ABA signal transduction (Zhu
et al. 2007). Previously, we found that CPK4 phosphorylates
ROP GUANINE NUCLEOTIDE EXCHANGE FACTOR
(RopGEF), a small GTP-binding protein exchange factor (Li
et al. 2018). Moreover, CPK4 promotes the degradation of
RopGEF in vacuoles via the endosome-prevacuolar
IN A NUTSHELL
Background: Plants experience osmotic stress during drought conditions and in soils with high salt levels. Osmotic
stress triggers a rapid rise in intracellular calcium ion (Ca
2+
) concentrations in plants. Upon the binding of Ca
2+
, the
calcium-dependent protein kinase (CPK) undergoes a conformational change, releasing the kinase active site from the
repression of an autoinhibitory junction. CPKs become active to phosphorylate their downstream targets to transduce
osmotic stress signals. CPK4 plays a positive role in plant osmotic stress responses.
Question: How are active CPK4 protein levels dynamically and precisely regulated in response to hyperosmotic stress?
Findings: We found that salt/mannitol-induced osmotic stress promotes CPK4 protein accumulation by disrupting
26S proteasome-mediated degradation of CPK4. The U-BOX E3 ligase PUB44 interacts with and mediates the ubiqui-
tination of CPK4, which results in the proteasomal degradation of CPK4. PUB44 exhibits a CPK4-dependent negative
role in plant osmotic stress responses. In addition, we found that Ca-binding and kinase activation decrease the ubi-
quitination of CPK4 and enhance CPK4 protein stabilization.
Next steps: We plan to investigate how the binding of Ca
2+
to CPK determines the ubiquitination and degradation of
CPK4.
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Figure 1. CPK4 regulates plant osmotic stress responses. A) Osmotic stress and ABA hormone treatments lead to CPK4 protein accumulation in
Arabidopsis whole seedlings. Ten-day-old ProUBQ:mCherry-CPK4-myc seedlings grown on ½ MS (half-strength Murashige–Skoog) medium were
incubated in ½ MS (half-strength Murashige–Skoog) liquid medium for 2 h then transferred into ½ MS (half-strength Murashige–Skoog) liquid
medium containing 300 mM mannitol, 150 mM NaCl, 50 µM ABA, 1 µM Auxin in a chamber with 21 °C, 120 μmol·m
−2
·s
−1
light for the indicated
times. For low/high-temperature treatments, seedlings in ½ MS (half-strength Murashige–Skoog) liquid medium was placed in the chamber with
4 °C or 37 °C, 120 μmol·m
−2
·s
−1
light. WT (Col-0) as a negative control. Total proteins from the whole seedlings were extracted and subjected to
SDS-PAGE gels. CPK4-myc protein level was detected by myc antibody and ponceau staining was used as a loading control. Note that about 20 µg
total proteins were loaded into SDS-PAGE gels. Note that immunoblots showing mannitol, NaCl, and ABA treatments were exposed for shorter
times (5 s) compared to other blots (30 s) in panel A, due to the observed increases in CPK4 protein levels. B) Mannitol-induced hyperosmotic
stress disrupts MG132-dependent degradation of CPK4. 10-day-old seedlings were treated with translation inhibitor 50 µM CHX, 50 µM CHX
plus 300 mM mannitol, or the 26 s proteasome inhibitor 50 µM MG132 for the indicated times. WT (Col-0) as a negative control. Total proteins
were isolated and subjected to IB analysis with myc antibody. Ponceau staining was used as a loading control. Note that about 20 µg total proteins
were loaded into SDS-PAGE gels. Immunoblots showing MG132 treatment were exposed for shorter times (5 s) compared to other blots (30 s) in
panel B, due to the observed increases in CPK4 protein levels. C) Seedling growth assays of WT, cpk4 cpk11, and CPK4 overexpression line (CPK4 OE)
under hyperosmotic conditions. Seeds of WT, cpk4 cpk11, and CPK4 overexpression line (CPK4 OE) collected at the same time were sterilized and
subsequently stratified for 5 days at 4 °C. Nine seeds of each genotype were directly spread and grown vertically on ½ MS (half-strength Murashige–
Skoog) medium supplemented with or without 300 mM mannitol. After 7-day growth, plates were photographed and root lengths were measured
(continued)
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compartment pathway (Li et al. 2018). ABA-induced
CPK-dependent degradation of RopGEFs reduces the negative
regulatory role of RopGEF in ABA signal transduction and
ABA-regulated lateral root growth (Li et al. 2018). To investigate
environmental factors that regulate CPK4 activity, we gener-
ated stable transgenic plants that overexpressed CPK4 and in-
vestigated the abundance of CPK4 protein under various
environmental conditions. Here, our findings describe the
PUB44-CPK4 signaling module mediates the response of plants
to osmotic stress. Osmotic stress down-regulates
PUB44-mediated degradation of CPK4 leading to CPK4 accu-
mulation to further activate osmotic stress response in plants.
Results
Hyperosmotic stress and ABA induces the
accumulation of CPK4
To investigate the environmental cues that control the activ-
ities of CPK4, stable transgenic lines overexpressing
mCherry-CPK4-myc driven by the POLYUBIQUITIN 10
(UBQ10) promoter were generated and CPK4 protein abun-
dance was investigated in response to several environmental
stimuli, including high/low temperature, mannitol/
NaCl-induced hyperosmotic stress, the phytohormone ABA
and auxin, and a deficiency of nitrate or phosphate nutrients.
Interestingly, we found that the exogenous application of
mannitol, NaCl, and ABA led to a rapid elevation of CPK4
protein abundance (Fig. 1A) while the levels of CPK4 protein
did not change substantially in response to high or low tem-
peratures, a lack of nitrate or phosphate nutrients, or auxin
treatments (Fig. 1A, Supplemental Fig. S1A). Considering
that CPK4 was driven by the constitutive UBQ10 promoter,
we suspect that changes in the levels of CPK4 protein may
be due to alterations in the stability of CPK4 protein instead
of different transcript levels. Consequently, we analyzed the
protein stability of CPK4 in the presence of cycloheximide
(CHX), a protein synthesis inhibitor, and found that CPK4
protein level gradually decreased (Fig. 1B). Interestingly, in
the presence of both CHX and mannitol, the levels of CPK4
protein remained stable over an 8-hour treatment (Fig. 1B).
Importantly, the application of MG132, an inhibitor of the
26S proteasome, induced CPK4 accumulation (Fig. 1B) and
the reduction of CPK4 protein levels in the presence of
CHX was compromised by the combination of MG132 and
CHX treatments (Supplemental Fig. S1B).
Next, we investigated the subcellular localization of CPK4. In
CPK4-stable Arabidopsis transgenic lines (Supplemental Fig.
S2A), fluorescence signals of Venus-CPK4 fusion proteins
were evident at the periphery and in the cytoplasm of
Arabidopsis root tip cells. Increased levels of Venus fluorescence
were detected as seedlings were exposed to ABA, NaCl,
mannitol-induced hyperosmotic stress, and MG132 treatment
(Supplemental Fig. S1C). These findings indicated that the levels
of CPK4 protein were controlled by an unknown ubiquitin lig-
ase that may be inhibited by osmotic stress treatment.
The accumulation of CPK4 protein under osmotic stress
conditions implies that CPK4 may modulate the osmotic
stress response in plants. Next, we analyzed seedling growth
and primary root elongation under hyperosmotic stress condi-
tions in cpk4 cpk11 double mutant and CPK4 overexpression
lines. cpk4 cpk11 double mutant plants exhibited enhanced
sensitivity to osmotic stress-repressed seedling growth and pri-
mary root elongation when compared to wild-type plants. The
osmotic stress-elicited phenotypes in the CPK4 overexpression
lines were indistinguishable from those of wild-type plants
(Fig. 1, C and D), although this CPK4 overexpression construct
complemented the cpk4 cpk11 mutant phenotype (Fig. 1E,
Supplemental Fig. S2, B and C). Furthermore, the exogenous
application of mannitol, NaCl, ABA, and MG132 elevated
the abundance of CPK4 protein in the complementation
line (Supplemental Fig. S2C). Taken together, these results in-
dicate that CPK4 plays a positive role in osmotic stress.
PUB44 interacted with CPK4 in vitro and in vivo
To search for potential ubiquitin E3 ligases that may promote
the degradation of CPK4, CPK4-myc seedlings were incu-
bated overnight with MG132 to reduce CPK4 degradation
and increase E3 ligase and CPK4 association.
Immunoprecipitation (IP) was then carried out and proteins
associated with CPK4-myc were identified by mass
(Figure 1. Continued)
by Image J software. The graph on the right shows a quantification of root length. Error bars show the mean ± Standard Error of Mean (SEM) of 3
independent replicates with 27 seedlings per replicate. A representative result from 3 independent experiments with similar results is shown.
Asterisks indicate statistically significant differences (***P < 0.001, Student’s t-test). Scale bar, 1 cm. D) Primary root elongation of WT, cpk4
cpk11 and CPK4 overexpression line (CPK4 OE) under hyperosmotic conditions. Three-day-old seedlings grown on ½ MS (half-strength
Murashige–Skoog) medium with similar primary root length were transferred on ½ MS (half-strength Murashige–Skoog) medium with or without
(control) 125 mM NaCl and grown on vertical plates. Representative photographs were taken after 6 days of growth (left). Root length was measured
using Image J software each day after the seedlings were transferred (right). Values are shown as mean ± SEM with 3 biological replicates in the
experiment and 30 seedlings for each biological replicate. Asterisks indicate statistically significant differences (*P < 0.05, Student’s t-test). Scale
bar, 1 cm. E) Expression of ProUBQ:mCherry-CPK4-myc in cpk4 cpk11 double mutant rescues the hypersensitive phenotype of cpk4 cpk11 to
NaCl-mediated inhibition of primary root growth. Two independent complemented lines (19#, 21#) of ProUBQ:mCherry-CPK4-myc cpk4 cpk11
(CPK4 Com) were screened out. Four-day-old seedlings grown on ½ MS medium with similar primary root length were transferred onto ½ MS plates
with or without 100 mM NaCl, after 7-day growth, plates were photographed and root length were measured by Image J software. The graph on the
right shows a quantification of root length. Error bars show mean ± SEM of 15 seedlings with 3 biological replicates in the experiment. Asterisks
indicate statistically significant differences (***P < 0.001, Student’s t-test). Scale bar, 1 cm.
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spectrometry (Supplemental Fig. S3A). A U-box type E3 ligase
(PUB44) was detected in 2 sequential IP-MS experiments.
PUB44, also referred to as SAUL1 serves as a positive regula-
tor of Pathogen Associated Molecular Pattern-triggered im-
munity (Tong et al. 2017). To test the interaction between
PUB44 and CPK4, we performed in vitro pull-down assays
using purified PUB44 and CPK4. Reciprocal pull-down assays
indicated that PUB44 bound physically to CPK4 (Fig. 2A).
Yeast 2-hybrid experiments showed that CPK4 had a weak
interaction with full-length PUB44 but exhibited a relatively
strong interaction with the Armadillo (ARM)-like domain of
PUB44 (Fig. 2B). PUB44 interacted with the CPK4 N terminus
and kinase domain while the autoinhibitory domain of CPK4
exhibited a negative role in CPK4 and PUB44 interaction
Figure 2. PUB44 associates with CPK4. A) In vitro pull-down assays show that CPK4 interacts with PUB44. His tagged CPK4-myc and GST tagged
PUB44 were purified from Escherichia coli, pull-down assays were conducted with Glutathione agarose beads or Nickel resin and the bound proteins
were eluted and analyzed by immunoblot probed with anti-myc or anti-GST antibody. IP, Immunoprecipitation. CBB, Coomassie Brilliant Blue stain-
ing. B) Yeast 2-hybrid assay shows that CPK4 preferentially interacts with PUB44 fragments with the ARM-like domain. The indicated constructs
were transformed into yeast (Saccharomyces cerevisiae) strain of PJ69-4ɑ. Overnight yeast cultures were diluted to an OD
600
= 1, then a series of
tenfold serial dilutions were spotted onto double (SD/L-T-) and triple (SD/L-T-H-) dropout selective medium. Photographs were taken at 3 days
(double) and 6 days (triple) after inoculation. 2.5 mM 3-AT (3-amino-1,2,4-triazole) was added to the medium to repress auto-activation.
Activation domain (AD)-CPK4 and (DNA-binding domain) DNA binding domain (BD)-RopGEF1 interaction was used as a positive control.
Activation domain (AD)-CPK4 and (DNA-binding domain) BD was used as negative control. The upper schematic depicts the predicted domain
of the PUB44 protein. C) Co-IP assays show the interactions between CPK4 and PUB44 in Arabidopsis. Left: CPK4 is associated with PUB44 instead of
PUB25. Arabidopsis plants co-expressing ProUBQ-mCherry-CPK4-myc and ProUBQ-Venus-PUB44-Flag or ProUBQ-mCherry-CPK4-myc and
ProUBQ-PUB25-Flag pub25 were generated through the crossing. Total proteins from 12-day-old seedlings were extracted and mixed with flag mag-
netic beads, and immunoprecipitates were detected with Flag and myc antibody. Right: NaCl treatment does not affect the association of CPK4 and
PUB44. Arabidopsis seedlings were incubated in ½ MS (half-strength Murashige–Skoog) solution with or without 150 mM NaCl for 4 h. Total pro-
teins were extracted and immunoprecipitated with Flag beads and then detected with anti-myc antibody. D) BiFC assays show that CPK4 associates
with PUB44 and ARM-like domain of PUB44 at cytosol and cell periphery. The indicted BiFC constructs were transiently co-expressed in Nicotiana
benthamiana leaves, and fluorescence was visualized through confocal microscopy. Using 5 rectangles of equal size to frame the cells and the fluor-
escence intensity values were exported by software. Average quantification values of Yellow Fluorescent Protein (YFP) signals are shown at the bot-
tom of the top panels (n = 5 images per combination). Scale bar, 50 µm.
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Figure 3. PUB44 ubiquitinates CPK4 in vitro and in vivo. A) In vitro kinase assays show that CPK4 does not phosphorylate PUB44. Recombinant
His-CPK4 was used to phosphorylate GST-PUB44 and control GST-RopGEF1 fusion proteins expressed and purified from Escherichia coli in the pres-
ence of
32
P-ATP. Autoradiograph (left) and Coomassie stain (CBB) (right) show phosphorylation and loading of purified CPK4 and PUB44.
Phosphorylation of RopGEF1 and histone type III-S embedded in SDS-PAGE were used to indicate the CPK4 activity. The asterisk indicates the pos-
ition of PUB44. Note that the blot shown in (A) is from the same gel as shown in Supplemental Fig. S4A. B) In vitro ubiquitination assays show that
Escherichia coli-derived PUB44 E3 ligase exhibits ubiquitin ligase activity. His-tagged E1 (AtUBA1), E2 (AtUBC8), and GST-tagged PUB44 were puri-
fied from Escherichia coli. The indicated proteins were incubated together for the ubiquitination reactions. PUB44 was detected by IB with a flag
antibody. C) In vitro ubiquitination assays show that PUB44 promotes CPK4 ubiquitination CPK4. Recombinant proteins purified from Escherichia
coli were incubated at 30 °C for 2 h to enable ubiquitination reactions. CPK4-myc protein was subsequently isolated from the mixture by IP using
myc antibody-conjugated magnetic beads and detected by IB with myc and ubiquitin antibody. The triangle indicates nonspecific bands. D) PUB44
autoubiquitination and ubiquitinates CPK4 in the bacterial ubiquitination system. The combination of PUB44-myc, AtUBA1 (E1), AtUBC8 (E2),
His-FLAG-Ub (Ubiquitin), and MBP-CPK4 were co-expressed in Escherichia coli. The bacterial lysates were subjected to IB analysis with anti-myc anti-
bodies to detect PUB44 autoubiquitination, and with anti-MBP antibodies to detect CPK4 ubiquitination. E) MS/MS spectra of the tryptic peptides
containing ubiquitinated K (lysine) 241 and K (lysine) 495 are shown. Gly-Gly (GG) is diglycine residue on ubiquitinated lysine residue. F) CPK4 2KR
(continued)
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(Supplemental Fig. S3B). Co-IP in transgenic Arabidopsis
seedlings confirmed that CPK4 associated with PUB44, but
not PUB25, and that NaCl-induced hyperosmotic stress
had no remarkable effect on the association between CPK4
and PUB44 (Fig. 2C). Furthermore, the association of CPK4
and PUB44 or the PUB44 ARM-like domain mainly occurred
in the cell periphery as observed in bimolecular fluorescence
complementation (BiFC) assays (Fig. 2D, Supplemental Fig.
S3, C and D). Collectively, these results indicate that PUB44
interacted with CPK4 in vitro and in vivo.
PUB44 ubiquitinated CPK4
Considering that CPK4 is a calcium-dependent protein kinase,
we first analyzed whether CPK4 phosphorylated PUB44. In vitro
kinase assays showed that purified recombinant CPK4 ex-
pressed in Escherichia coli exhibited kinase activity toward
RopGEF1(Li et al. 2018) but not PUB44 (Fig. 3A,
Supplemental Fig. S4A). To test whether PUB44 ubiquitinates
CPK4, we performed in vitro ubiquitination assays using
His-tagged Arabidopsis E1 protein (AtUBA1) as well as E2
(AtUBC8) and Glutathione S-transferase (GST)-tagged PUB44
and CPK4-myc purified from Escherichia coli. PUB44 exhibited
strong E3 ubiquitin ligase activity and could add ubiquitin to
CPK4 as shown in protein ladder-like immunoblot signals
(Fig. 3, B and C). Similarly, we detected the autoubiquitination
of PUB44 and ubiquitination of CPK4 by PUB44 in a bacterial
ubiquitin system (Fig. 3D) (Han et al. 2017).
Furthermore, maltose binding protein (MBP)-CPK4 pro-
tein was isolated from bacteria that co-expressed E1, E2,
PUB44, and CPK4 with dextrin beads and subjected to tan-
dem spectrometry analyses. Two lysine residues, lysine 241
and 495, were identified as ubiquitination sites. Combined
mutations of lysine 241 and 495 to arginine (2KR) compro-
mised PUB44-mediated CPK4 ubiquitination (Fig. 3, E and F).
Taken together, these experimental data suggested that
PUB44 ubiquitinated CPK4 in vitro.
To investigate whether CPK4 is ubiquitinated in plants,
CPK4-myc was isolated from the total protein of
mCherry-CPK4-myc seedlings and analyzed with myc and
ubiquitin antibodies. Immunoblotting (IB) detected a band
with a higher predicted molecular weight (MW) of CPK4
when using the myc antibody and a smear corresponding
to purified CPK4 protein using the ubiquitin antibody
(Fig. 3G). Native PAGE gels, Phos-tag gels, and Calf
Intestinal Alkaline Phosphatase treatment experiments indi-
cated that this higher predicted MW band of CPK4 was not a
dimer or phosphorylated-form of CPK4 (Supplemental Fig.
S4, B to D). These experiments suggested that CPK4 is ubiqui-
tinated in Arabidopsis.
Similarly, we enriched ubiquitinated proteins using
TUBE2-conjugated beads and detected the ubiquitination
of CPK4-myc with the myc antibody. This experiment con-
firmed that CPK4 is ubiquitinated in Arabidopsis (Fig. 3H).
To test whether CPK4 ubiquitination in plants is mediated
by PUB44, mCherry-CPK4-myc was introduced into the
pub44 mutant and Venus-PUB44-Flag overexpressing plants
by crossing. Near equal amounts of immuno-precipitated
CPK4-myc protein were isolated from these different geno-
types, as detected by the myc antibody (Fig. 3I). IB results
showed that the levels of ubiquitinated CPK4-myc were re-
duced in pub44 mutant plants (Fig. 3, I and J). Taken together,
these analyses provide evidence that PUB44 can promote
CPK4 ubiquitination in vitro and in vivo.
PUB44 promoted the degradation of CPK4 protein
To investigate the role of PUB44 in the stability of CPK4 pro-
tein, we crossed mCherry-CPK4-myc Col (CPK4 OE) with the
pub44 mutant and Venus-PUB44-Flag (PUB44 OE) overex-
pression lines. Then, we investigated the levels of CPK4 pro-
tein in CPK4 OE Col, CPK4 OE pub44, and CPK4 OE PUB44 OE
plants by IB. We found that the co-expression of PUB44 and
CPK4 led to reduced levels of CPK4 protein while CPK4 OE
pub44 plants exhibited increased levels of CPK4 protein in
the root and leaf tissues and whole seedlings of
Arabidopsis (Fig. 4, A and B, Supplemental Fig. S5A).
Reverse transcription-quantitative PCR analysis indicated
that CPK4 transcripts were comparable in CPK4 OE Col,
CPK4 OE pub44, and CPK4 OE PUB44 OE plants (Fig. 4C),
(Figure 3. Continued)
(K241RK495R, combined mutations of lysine 241 and 495 to arginine) shows compromised PUB44-mediated ubiquitination. The combination of
PUB44-myc, AtUBA1 (E1), AtUBC8 (E2), His-FLAG-Ub (Ubiquitin), and MBP-CPK4 or MBP-CPK4 K241/495R were co-expressed in Escherichia coli.
The bacterial lysates were subjected to IB analysis with anti-myc antibodies to detect PUB44 autoubiquitination, and with anti-MBP antibodies to
detect CPK4 ubiquitination. G) CPK4 is ubiquitinated in vivo. 10-day-old mCherry-CPK4-myc seedlings were treated with 50 µM MG132 for 4 h.
Total protein was extracted and IP was performed using myc antibody-conjugated magnetic beads. CPK4-myc was detected with myc and ubiquitin
antibody. H) CPK4 is ubiquitinated in vivo. Ten-day-old mCherry-CPK4-myc seedlings were treated with 50 µM MG132 for overnight. Ubiquitinated
proteins were enriched by TUBE2 coupled magnetic beads and immunoprecipitates (IB) were detected with myc and ubiquitin antibody. I and J)
CPK4 ubiquitination in pub44 and PUB44 overexpression lines (PUB44 OE) compared with wild-type (WT) plants. mCherry-CPK4-myc was intro-
duced into pub44 mutant and Venus-PUB44-Flag overexpression (PUB44 OE) plants through crossing. Ten-day-old seedlings of each genotype
were treated with 50 µM MG132 for 4 h. Total protein was extracted and CPK4-myc protein was isolated with myc antibody-conjugated magnetic
beads. Approximately equal amount of immunoprecipitated (IP) CPK4-myc was loaded into SDS-PAGE and the ubiquitination of CPK4-myc was
detected with anti-ubiquitin antibodies (I). The ubiquitination levels of CPK4 were quantified with ImageJ software. The extent of CPK4 ubiquitina-
tion is calculated as (CPK4-Ub intensity)
IP
/(CPK4-myc intensity)
IP
. The extent of Ub
n
-CPK4 in pub44 and PUB44 OE background was normalized
against that of CPK4 OE Col0 plants that were set to 1.0 (J). Data are means ± SEM of 3 independent biological replicates. Asterisks represent sig-
nificant differences compared to the CPK4 OE Col0 (**P < 0.01, Student’s t-test).
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indicating a negligible contribution of change in CPK4 tran-
scripts to the altered levels of CPK4 protein.
Next, we investigated the stability of CPK4 protein in the
presence of CHX. We observed a gradual degradation of
CPK4 protein in CPK4 OE Col plants in the presence of
CHX (Figs. 4D and 1B, CHX). Comparatively, CPK4 protein le-
vels did not vary notably in CPK4 OE pub44 plants in the pres-
ence of CHX (Fig. 4D, CHX). Furthermore, the levels of CPK4
protein in CPK4 OE PUB44 OE plants were low and appeared
to remain at a persistently low level across the 4-hour CHX
treatment (Fig. 4D, CHX). In addition, the application of
MG132 or NaCl induced the accumulation of CPK4 in both
CPK4 OE Col and CPK4 OE pub44 plants, thus suggesting
the existence of other ubiquitin ligases that can target
CPK4 for degradation (Fig. 4D, MG132, NaCl). We also found
that the MG132- or NaCl-induced accumulation of CPK4
protein was rapid and substantial in CPK4OE PUB44OE lines,
which indicated that the low levels of CPK4 protein in
CPK4OE PUB44OE plants could be attributed to the PUB44
mediated ubiquitination and degradation of CPK4 (Fig. 4D,
MG132, NaCl). Noting that the double band of CPK4, (occa-
sional small size band) appeared in some immunoblots may
be a result of nonspecific recognition of CPK4-myc by the
myc antibody.
We also immunoprecipitated transgenic wild type (WT)
seedlings expressing CPK4-myc treated with or without
Figure 4. PUB44 promotes the degradation of CPK4. A and B) CPK4 protein levels in the indicated genotypes. Total proteins extracted from root or
leaf tissue of 12-day-old seedlings of mCherry-CPK4-myc WT(Col-0), mCherry-CPK4-myc pub44, and mCherry-CPK4-myc Venus-PUB44-Flag lines were
subjected to IB analysis. CPK4-myc was detected with myc antibody and tubulin was used as a loading control (A and B). Quantification of CPK4
protein level with ImageJ software. The band signal was first normalized against tubulin (myc band intensity)/(tubulin band intensity) and relative
protein level was calculated as value against the mCherry-CPK4-myc WT (Col-0) that was set to 1.0 (A and B). Data are means ± SEM of 3 independ-
ent biological replicates. Asterisks represent significant differences compared to the mCherry-CPK4-myc WT (Col-0) (***P < 0.001, Student’s t-test).
C) Reverse transcription quantitative PCR (RT-qPCR) analysis of the CPK4 transcripts in the plants from (A). Total RNAs from 10-day-old seedlings
were used for RT-qPCR analysis. The gene expression levels of CPK4 were normalized to ACTIN and presented as values relative to that in WT back-
ground. Each bar represents the mean ± SEM values of 3 replicates. D) Stability of CPK4-myc is enhanced in the absence of PUB44. 10-day-old
mCherry-CPK4-myc Col, mCherry-CPK4-myc pub44, and mCherry-CPK4-myc Venus-PUB44-Flag seedlings were incubated in ½ MS (half-strength
Murashige–Skoog) liquid medium containing 50 µM CHX, 100 µM MG132 or 150 mM NaCl for the indicated time. Total proteins were extracted
and subjected to SDS-PAGE. CPK4-myc was detected with myc antibody and tubulin was used as a loading control. The experiments were repeated
at least twice with similar results. E and F) NaCl or mannitol-induced hyperosmotic stress decreases CPK4 ubiquitination. E) Ten-day-old
mCherry-CPK4-myc Col seedlings were treated with 150 mM NaCl or 300 mM mannitol for 4 h. Total proteins were extracted and almost equal
amount of total protein was immunoprecipitated (IP) with myc antibody-conjugated magnetic beads. CPK4-myc was detected by IB with anti-myc
and anti-ubiquitin antibodies. F) Relative ubiquitination levels were quantified with Image J software. The extent of CPK4 ubiquitination is calculated
as (CPK4-Ub intensity)
IP
/(CPK4-myc intensity)
IP
. Relative ubiquitination level was calculated as value against that in mock-treated plants that were
set to 1.0. Data are means ± SEM of 3 independent biological replicates. Asterisks represent significant differences compared to the mock-treated
plants. WT(Col-0) as a negative control. (**P < 0.01, ***P < 0.001, Student’s t-test).
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NaCl or mannitol to investigate whether hyperosmotic stress
exerted an influence on CPK4 ubiquitination and whether
any alteration in CPK4 ubiquitination could account for
the hyperosmotic stress-induced accumulation of CPK4 pro-
tein. Immunoblot analyses showed that near equal amounts
of immunoprecipitated proteins from NaCl and mannitol-
treated samples exhibited less CPK4 ubiquitination than
those from mock-treated samples (Fig. 4, E and F,
Supplemental Fig. S5, B and C). The decrease in CPK4
ubiquitination under NaCl/mannitol treatment could be
partially restored by combined MG132 and NaCl/mannitol
treatments (Supplemental Fig. S5C), thus suggesting that
ubiquitinated CPK4 underwent degradation via the 26S pro-
teasome. These experiments suggested that osmotic stress
induced the accumulation of CPK4 protein by reducing
CPK4 ubiquitination. In summary, our data suggest that
PUB44 mediated the destabilization of CPK4 via the 26S
proteasome-dependent pathway.
Figure 5. PUB44 plays a negative role in osmotic stress-induced root growth inhibition. A and B) pub44 mutant and overexpression of
Venus-PUB44-Flag (PUB44OE) plants exhibited reduced or increased sensitivity to hyperosmotic stress in root growth respectively compared
with WT. Four-day-old seedlings of each genotype grown on ½ MS medium with similar root length were transferred onto ½ MS (half-strength
Murashige–Skoog) medium with 300 mM mannitol. Representative photographs were taken after 6 days growth (A) and root length was measured
using Image J software each day after the seedlings were transferred (B). Values are shown as mean ± SEM, with 3 biological replicates in the ex-
periment and 30 seedlings for each biological replicate (**P < 0.01, Student’s t-test). Scale bar, 1 cm. C and D) Root elongation of indicated genotypic
plants grown on the medium with 300 mM mannitol. Four-day-old seedlings grown on ½ MS (half-strength Murashige–Skoog) medium with similar
root lengths were transferred onto ½ MS (half-strength Murashige–Skoog) medium with 300 mM mannitol. Representative photographs were taken
(C) and root length was quantified (D) using image J software 7 days after the seedlings were transferred. Values are shown as mean ± SEM, with 3
biological replicates in the experiment and 15 seedlings for each biological replicate (*P < 0.05, ***P < 0.001, Student’s t-test). Scale bar, 1 cm. E and
F) 3,3′-DAB staining of primary roots of the PUB44 overexpression line (PUB44 OE), WT, and pub44 mutant plants. Five-day-old seedlings grown on ½
MS (half-strength Murashige–Skoog) medium were transferred onto ½ MS (half-strength Murashige–Skoog) liquid medium treated with 300 mM
mannitol for 8 h and then subjected to DAB staining (E). The relative intensity of DAB staining in (F) was measure with ImageJ software. The intensity
of WT was set to 1.0. Values are shown as mean ± SEM, with 3 biological replicates in the experiment and 10 seedlings for each biological replicate
(*P < 0.05, ***P < 0.001, Student’s t-test). Scale bar, 100 µm. G and H) 3,3′-DAB staining of the primary roots of the WT, pub44, cpk4 cpk11, and
pub44 cpk4 cpk11 triple mutant treated with 300 mM Mannitol for 8 h (G). The relative intensity of 3,3′-DAB staining in (H) was measure with
ImageJ software. The intensity of WT was set to 1.0. Values are shown as mean ± SEM, with 3 biological replicates in the experiment and 10 seedlings
for each biological replicate (**P < 0.01, ***P < 0.001, Student’s t-test). Scale bar, 100 µm. I) Proline content in the roots of WT, pub44, cpk4 cpk11,
and pub44 cpk4 cpk11. Twelve-day-old seedlings grown on ½ MS (half-strength Murashige–Skoog) medium were transferred into ½ MS (half-
strength Murashige–Skoog) liquid medium treated with or without 300 mM mannitol for 8 h and then roots were cut and harvested to measure
proline content. Values are means ± SEM with 3 biological replicates in the experiment (n = 3). (*P < 0.05, ***P < 0.001, Student’s t-test).
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Figure 6. Osmotic stress reduced PUB44 E3 ligase activity. A) GUS staining of transgenic Arabidopsis plants carrying the ProPUB44:GUS construct in the
presence or absence of mannitol treatment. Five-day-old seedlings grown on ½ MS (half-strength Murashige–Skoog) medium were transferred onto ½
MS (half-strength Murashige–Skoog) liquid medium treated with or without 300 mM mannitol for the indicated time and then subjected to GUS
staining. Scale bar, 1 cm. B and C) Subcellular localization of PUB44 with or without NaCl treatment. Five-day-old transgenic seedlings were transferred
into liquid 1/2 MS (half-strength Murashige–Skoog) with or without 150 mM NaCl. After 30 min treatment, the confocal images of root tip region were
taken by a LSM 900 microscope (Carl Zeiss). Microscopy and imaging parameters were identical for the images in a figure. The green fluorescence of
confocal images indicates Venus signal (B). Boxplots of the quantification of fluorescence intensity in the indicated treatments (C). Boxplot elements:
center line, median; box limits, upper and lower quartiles; whiskers, 1.5 × interquartile range. Each dot represents an independent root fluorescence
intensity. The relative fluorescence intensity values of root cells were measured and exported by Zeiss ZEN 3.4 software. Fluorescence intensity values in
the graphics correspond to the means ± SEM of 15 independent samples. n.s. indicates no significant differences. Scale bar, 20 µm. D) Immunoblot
analyses showed that osmotic stress did not cause the accumulation of PUB44 protein. Ten-day-old Venus-PUB44-Flag seedlings were treated with
½ MS (half-strength Murashige–Skoog) liquid medium containing 150 mM NaCl or 300 mM mannitol (Man) for the indicated times. Total proteins
were isolated and subjected to IB analysis. PUB44-Flag was detected with Flag antibody. Tubulin was used as a loading control. E and F) Luciferase
complementation imaging (LCI) assays of CPK4 and PUB44 interaction with or without 150 mM NaCl treatment. PUB44-nLuc and cLuc-CPK4
were co-expressed in Nicotiana benthamiana leaves. cLuc-CPK12 was used as a negative control (E). The luciferase activity was measured using
Promega-GloMax 20/20 Luminometer (F). Quantification of luminescence intensities in the indicated treatments. Luminescence intensity acquired
from fifteen Nicotiana benthamiana leaves was used to calculate the average luciferase activity. Values are means ± SEM (n = 15), n.s. indicates not
significant differences. nLuc, N-terminal luciferase. cLuc, C-terminal luciferase. The color scale represents luciferase activity levels. Scale bar, 1 cm. G
and H) Quantitative BiFC of CPK4 and PUB44 interaction with or without 150 mM NaCl treatment. Yellow Fluorescent Protein (YFP)
N
-CPK4 and
YFP
C
-PUB44 were co-transformed in Nicotiana benthamiana leaves. 48 h after transformation, sterilized water or 150 mM NaCl was injected into leaves.
Left: Confocal images are presented and the YFP fluorescence indicates interaction signal (G). Right: the quantification of fluorescence intensity in the
indicated treatments (H). Microscopy and imaging parameters were identical for the images. The relative fluorescence intensity was measured by Zeiss
ZEN 3.4 software. Using 5 rectangles of equal size to frame the cells and the fluorescence intensity values were exported by software. Fifteen independ-
ent photographs were used to calculate the average fluorescence intensity. Values are means ± SEM (n = 15), n.s. indicates not significant differences.
(continued)
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PUB44 contributed to hyperosmotic stress sensitivity
To investigate the biological relevance of CPK4 and PUB44,
we generated stable transgenic plants overexpressing
PUB44 driven by the constitutive UBQ promoter
(Supplemental Fig. S2D). Next, pub44 mutant
(Supplemental Fig. S2E) and PUB44 overexpression lines
were subjected to an osmotic stress tolerance assay.
Compared with wild-type plants, pub44 mutant, and
PUB44 OE plants possessed longer or shorter primary roots,
respectively, when grown on a mannitol-containing medium
(Fig. 5, A and B). Under control conditions, the pub44 mutant
and PUB44 overexpression lines showed similar primary root
(Figure 6. Continued)
The photographs were taken after 30 min NaCl treatment using a LSM 900 microscope (Carl Zeiss). Scale bar, 50 µm. I and J) Effect of osmotic stress
on PUB44 E3 ligase activity. Ten-day-old ProUBQ-Venus-PUB44-Flag and WT nontransgenic seedlings were treated with 150 mM NaCl or 300 mM
mannitol for 4 h. Total protein was extracted and immunoprecipitated with Flag antibody-conjugated magnetic beads. PUB44-Flag was detected by
IB with anti-Flag or anti-ubiquitin antibody (I). Relative ubiquitination level were quantified with Image J software. The extent of PUB44 ubiquitina-
tion is calculated as (PUB44-Ub intensity)
IP
/(PUB44-Flag intensity)
IP
. The extent of Ub
n
-PUB44 in NaCl and Mannitol-treated plants was normalized
against that of mock plants that was set to 1.0 (J). Values are means ± SEM with 3 biological replicates in the experiment (n = 3) (*P < 0.05,
***P < 0.001, Student’s t-test).
Figure 7. Inactive-form CPK4 undergoes preferential degradation. A) Protein levels of various CPK4 variants. Total protein was extracted from
10-day-old homozygous transgenic seedlings overexpressing CPK4-myc, CPK4-CA-4myc (the constitutively active form of CPK4),
CPK4-D149A-myc (the kinase-dead version of CPK4), or CPK4-EF-4myc (the EF-hand mutated version of CPK4, CPK4-EF is generated through mu-
tation of conserved Asp to Ala in EF hand sequences (EF1: D339, 341A, EF2: D375, 377A, EF3: D411, 413A, and EF4: D445,447A). Total protein was
subjected to SDS-PAGE and detected with myc antibody. Tubulin was used as an internal control. B) Reverse transcription quantitative PCR
(RT-qPCR) analysis of the CPK4 transcripts in the plants from A. Total RNAs from 10-day-old seedlings were used for RT-qPCR analysis. Each
bar represents the mean ± SEM values of 4 replicates. The dots indicate individual data points. The gene expression levels of CPK4 were normalized
to ACTIN and presented as values relative to that in wild-type (WT) background. C and D) The ubiquitination levels of various CPK4 variants.
Ten-day-old seedlings of each genotype were treated with 50 µM MG132 for 4 h. Total protein was extracted and quantified. Approximately equal
amount of total protein was incubated with myc antibody-conjugated magnetic beads. Immunoprecipitated (IP) myc tagged CPK4 variants were
loaded into SDS-PAGE and detected by IB with myc and ubiquitin antibodies. Tubulin was used as an internal control.
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lengths as those in wild-type plants when grown on a normal
MS medium (Supplemental Fig. S6, A to D). These analyses
suggested that PUB44 plays a negative role in primary root
growth inhibition induced by osmotic stress.
Interestingly, a previous study showed that heterozygous
PUB44 pub44 plants exhibited excellent germination rates
while wild-type seeds exhibited strong germination repres-
sion in the presence of mannitol and NaCl (Salt et al.
2011). In addition, we observed that the young leaves of
pub44 mutant plants turned yellow after 7 days of growth
on MS medium containing mannitol (Fig. 5, A and C). This
leaf yellowing phenotype might be attributed to the en-
hanced auto-immunity when seedlings grew in normal con-
ditions at 21 °C, as reported previously (Disch et al. 2016).
Furthermore, we measured osmotic stress-triggered ROS
and the accumulation of proline. We found that the root tis-
sues of pub44 mutant and PUB44OE plants exhibited lower
and higher levels of ROS accumulation, respectively, when
compared with wild-type root tissue (Fig. 5, E and F).
Conversely, the levels of proline in the root tissue of the
pub44 mutant were higher than those in wild-type controls
(Fig. 5I). These phenotypic assays suggested that PUB44
acts as a negative regulator of the osmotic stress response.
We further generated pub44 cpk4 cpk11 triple mutants by
crossing and checking primary root elongation, ROS levels,
and proline content in WT, pub44 mutant, cpk4 cpk11 dou-
ble mutant, and pub4 cpk4 cpk11 triple mutant lines under
hyperosmotic conditions. Longer primary roots were ob-
served in the pub44 mutant under hyperosmotic conditions
(Fig. 5, A to D). Interestingly, the primary root length of the
pub44 cpk4 cpk11 triple mutant was similar to that of cpk4
cpk11 double mutant plants, thus suggesting that hyposensi-
tivity to osmotic stress-repressed primary root growth in the
pub44 mutant was dependent on CPK4 and CPK11 (Fig. 5, C
and D). In addition, pub44 and cpk4 cpk11 plants exhibited
lower and higher levels of ROS accumulation than those of
wild-type plants under hyperosmotic conditions. However,
pub44 cpk4 cpk11 resembled cpk4 cpk11 in terms of hyperos-
motic stress-induced ROS accumulation (Fig. 5, G and H).
Similarly, the levels of proline were consistently lower in
pub44 cpk4 cpk11 and cpk4 cpk11 mutants than in the WT.
In contrast, pub44 mutant plants exhibited a constitutively
higher level of proline than wild-type plants (Fig. 5I). These
results suggested that PUB44 acts genetically upstream of
CPK4 to negatively modulate the response to osmotic stress.
Hyperosmotic stress down-regulated PUB44 E3 ligase
activity
Considering that osmotic stress induced the accumulation of
CPK4 protein and that PUB44 promoted CPK4 ubiquitina-
tion and degradation, we hypothesized that osmotic stress
may elevate the levels of CPK4 protein, potentially by con-
trolling the behavior of PUB44 on CPK4. First, we examined
the transcription and protein stability of PUB44 under hyper-
osmotic conditions in stable homozygous ProPUB44:GUS and
ProUBQ:Venus-PUB44-Flag transgenic lines. GUS staining
showed that NaCl- or mannitol-induced osmotic stress
strongly induced the expression of PUB44 in the leaves and
roots of seedlings grown on MS medium or in soil (Fig. 6A,
Supplemental Fig. S7A). The co-expression of CPK4 and
PUB44 in seedlings and roots was supported by public gene
expression data sets (Supplemental Fig. S8A, B).
Next, we investigated the subcellular localization of
Venus-PUB44 in the presence and absence of osmotic stress.
Venus-PUB44 fluorescence was evident in the periphery of
cells, as demonstrated by colocalization with FM4-64, a dye
that stains the membrane of cells (Supplemental Fig. S7B).
The cellular localization of Venus-PUB44 was not substantial-
ly affected under hyperosmotic stress (Fig. 6, B and C).
Similarly, we did not observe notable PUB44 protein degrad-
ation under mannitol- or NaCl-induced osmotic stress
(Fig. 6D).
Furthermore, we investigated the effect of osmotic stress
on the interaction of PUB44 and CPK4. Luciferase comple-
mentation imaging (LCI) assays indicated that PUB44 inter-
acted with CPK4 instead of CPK12 and that NaCl
treatment exerted a negligible effect on CPK4 and PUB44
interaction (Fig. 6, E and F). BiFC assays showed that the ef-
fect of NaCl treatment on CPK4 and PUB44 interaction was
not statistically significant (Fig. 6, G and H); these results were
similar to those generated by Co-IP assays (Fig. 2C). Finally,
we determined the E3 ubiquitin ligase activity of PUB44 in
the absence or presence of hyperosmotic treatment; lower
autoubiquitination signals were detected from immuno-
precipitated PUB44-Flag from NaCl or mannitol-treated
seedlings than that of mock-treated seedlings (Fig. 6, I and
J, Supplemental Fig. S7C), thus suggesting that osmotic stress
potentially represses the activity of PUB44 ubiquitin ligase. In
addition, we mapped a mannitol treatment-enriched phos-
phorylation site in the U-box domain of PUB44 by mass spec-
trometry analysis. A phospho-mimicking mutation of PUB44
Threonine 77 (T77E) to Glutamic acid substantially reduced
the ubiquitin ligase activity of PUB44 as shown in bacteria
ubiquitination experiments (Supplemental Fig. S7D).
An inactive-form of CPK4 underwent preferential
degradation
The binding of Ca
2+
to the EF-hand motif of CPK triggers its
conformation change, thus releasing auto-inhibition on ki-
nase activity; subsequently, CPK4 is self-phosphorylated
and activated. While studying osmotic stress-induced CPK4
protein accumulation, we also investigated the effects of os-
motic stress on the protein levels of CPK4-D149A (a kinase-
dead version of CPK4) (Li et al. 2018), CPK4-EF (mutations of
key amino acids responsible for Ca
2+
binding in all 4 EF-hand
motifs) (Franz et al. 2011) and CPK4-CA (constitutively ac-
tive form of CPK4) (Boudsocq et al. 2010) in stably trans-
formed Arabidopsis lines. We found that the protein levels
of CPK4-D149A and CPK4-EF were extremely low compared
to that of CPK4, while the level of CPK4-CA was obviously
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higher than that of CPK4 (Fig. 7A), although all 4 transgenic
plants show similar CPK4 transcript levels (Fig. 7B).
Intriguingly, the CPK4-D149A protein level remained at a
low level and did not show any noticeable reduction during
8-h CHX treatments (Supplemental Fig. S9A), and did not
show a clear increase under NaCl and mannitol treatments
(Supplemental Fig. S9A). In addition, CPK4-EF, which re-
sembled CPK4-D149A, did not exhibit a reduction of pro-
tein level in response to CHX, and protein accumulation
under NaCl and mannitol treatments (Supplemental
Fig. S9B). Conversely, CPK4-CA behaved similarly to
CPK4 in response to CHX-mediated protein degradation
and NaCl/mannitol-induced protein accumulation
(Supplemental Fig. S9C).
Based on these results, we hypothesized that CPK4-D149A
and CPK4–EF undergo constitutive ubiquitination and degrad-
ation, therefore leading to low protein levels. Next, we mea-
sured the ubiquitination levels of CPK4, CPK4-D149A,
CPK4-EF, and CPK4-CA and found that the ubiquitination le-
vels of CPK4-D149A and CPK4-EF were substantially enhanced
when compared with CPK4 (Fig. 7, C and D). However,
CPK4-CA exhibited higher protein levels and a relatively low le-
vel of ubiquitination. These analyses provide initial evidence
that the inactive form of CPK4 was preferentially degraded.
Discussion
How activated CPKs are regulated is an important subject for
plant stress signaling. In this study, we found that osmotic
stress and ABA increase the protein level of CPK4 and iden-
tified a U-Box E3 ligase PUB44 that directly regulates the sta-
bility of CPK4 protein. Several results support this conclusion.
First, PUB44 interacted with CPK4 in both yeast and plants.
Second, PUB44 ubiquitinated CPK4 in vitro and the levels
of ubiquitinated CPK4 in vivo changed in line with the ex-
pression of PUB44. Third, CPK4 protein degradation was dis-
rupted in pub44 mutant, and the accumulation of CPK4
protein in response to osmotic stress was accelerated in
plants that overexpressed PUB44. Moreover, we observed
the reduced ubiquitin ligase activity of PUB44 in the presence
of osmotic stress, thus providing evidence that a reduction in
PUB44 activity contributed to osmotic stress-induced CPK4
protein accumulation.
CPK4 has been reported to function as a key regulator in
plant pathogen defense, drought tolerance, and ABA signal
transduction, and activates the ABRE-BINDING Factor 2
(ABF2) transcription factor (Zhu et al. 2007; Boudsocq
et al. 2010). Interestingly, our results showed that CPK4 pro-
tein levels increased substantially in response to mannitol/
NaCl-induced hyperosmotic stress, but not to environmental
temperature changes and nitrate or phosphate deficiency.
Emerging evidence supports the existence of crosstalk be-
tween osmotic stress signal transduction and defense me-
chanisms against pathogens. For example, BONZAI
proteins regulate plant immune responses and control global
osmotic stress responses in Arabidopsis (Chen et al. 2020).
PUB44, also referred to as SAUL1, plays an essential role in
natural plant immunity (Tong et al. 2017). Our results dem-
onstrate a functional link between the CPK4 protein and
PUB44 in modulating the plant osmotic stress response.
Unlike CPK4, which promotes plant responses to osmotic
stress, PUB44 plays a negative role in osmotic stress-triggered
primary root growth inhibition, ROS, and proline accumula-
tion. However, mannitol/NaCl-induced hyperosmotic stress
enhanced the transcription levels of PUB44, which may con-
tribute to establishing a negative feedback loop to modulate
the duration and intensity of stress responses. Raab et al
showed that PUB44 (SAUL1) targeted Arabidopsis
ALDEHYDE OXIDASE 3(AAO3), a key enzyme responsible
for ABA biosynthesis for degradation, and ABA levels were
enhanced in pub44 mutants (Raab et al. 2009). It is likely
that ABA signaling might exert a negative influence on
PUB44 E3 ligase activity. Therefore, the exogenous applica-
tion of ABA could impair the PUB44-mediated degradation
of CPK4 and increase the protein level of CPK4. Notably,
the leaves of the pub44 mutant became yellow under hyper-
osmotic conditions; this may be attributable to the enhanced
auto-immunity in the pub44 mutant (Disch et al. 2016) un-
der our growth conditions.
It is also notable that excessive activation of CPK4 may ex-
ert negative effects on plant growth as observed in curled
leaves and dwarf plantlets upon overexpression of a constitu-
tively active form of CPK4 (CPK4-CA) (Supplemental Fig. S2F).
Therefore, the abundance of activated CPK4 might be tightly
controlled by the PUB44-mediated ubiquitin-26S proteasome
degradation pathway under nonstressful growth conditions.
Arabidopsis CPK4 exhibits half maximal kinase activity in the
presence of approximately 3 μM of free Ca
2+
. However, under
normal conditions, plant cells retain a relatively low concentra-
tion of free cytosolic calcium (around 100 nM). Most of the
CPK4 proteins may remain in a calcium-free and inactive state
that can be preferentially degraded, thus maintaining the low
level of the active-form of CPK4.
CPKs play essential roles in plant responses to abiotic/biotic
stress. To enhance CPK4 protein levels during the response to
osmotic stress, the transcriptional of CPK4 is increased.
Hyperosmotic stress-triggered intracellular calcium rise leads
to the increase of the active-form of CPK4. Furthermore, hyper-
osmotic stress down-regulates the PUB44 E3 ligase activities.
Therefore, the PUB44-mediated degradation of CPK4 is inhib-
ited. Interestingly, we found that the phosphorylation of threo-
nine 77 in the U-box domain of PUB44 was enriched under
conditions of hyperosmotic stress. A phospho-mimicking mu-
tation of PUB44 T77E greatly reduced the ubiquitin ligase activ-
ity of PUB44. The interaction between U-box type E3 ligases
and kinases is a common theme. CPK28 mediated T95 phos-
phorylation on PUB25 to enhance PUB25 E3 ligase activity
(Wang et al. 2018). Similarly, the phosphorylation of Thr62/
88 by MPK3 resulted in a marked increase in the stabilization
and E3 ligase activity of PUB22 (Furlan et al. 2017).
Identification of the relevant kinases responsible for the
hyperosmotic stress-triggered phosphorylation of PUB44
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could help to clarify the molecular mechanisms underlying
the hyperosmotic stress-mediated repression of PUB44 E3
ligase activity. The PUB44-mediated control of CPK4 protein
abundance could provide the advantage of a more rapid and
tunable adjustment to balance growth and responses to the
fluctuating natural environment. In addition, osmotic stress
induced a rapid increase in intracellular calcium and the ac-
tivation and stabilization of CPK4, thus indicating that the
change in CPK4 protein stabilization may represent a mech-
anism that regulates signaling on this time scale. We assume
that CPK4 stabilization may be involved in longer-term accli-
mation and response considering the existence of extensive
downstream targets of CPK4 (Zhu et al. 2007).
Another unexpected and interesting observation in the
present study is that inactive variants of CPK4, including kinase-
dead CPK4-D149A and calcium-free CPK4-EF, were preferen-
tially degraded. Extensive studies have demonstrated that
CPK auto-phosphorylation occurs in a Ca
2+
-dependent man-
ner. Our previous analyses confirmed that the kinase activity
of CPK4 is activated by elevated levels of Ca
2+
(Li et al. 2018).
When Ca
2+
binds to the EF-hands in the CaM-like domain,
the CPK protein undergoes a conformational change, thus, re-
leasing the kinase active site from the repression of an autoin-
hibitory domain. Interestingly, in yeast 2-hybrid assays, PUB44
recognized the CPK4 N-terminus and kinase domain, and the
autoinhibitory domain repressed the binding of PUB44 to the
CPK4 kinase domain (Supplemental Fig. S3B). In addition, 2 ly-
sine residues were identified as ubiquitination sites in our mass
spectrometry analyses. Lysine 241 is located in the kinase do-
main and next to the autoinhibitory domain; lysine 495 is lo-
cated in the C terminus and adjacent to the EF-hand motif.
It is possible that a conformational change of CPK4 caused
by calcium binding would affect the accessibility of ubiquitina-
tion sites to those catalytic enzymes responsible for the ubiqui-
tination reaction. Further structural biology studies will
enhance our understanding of the role of Ca
2+
and the Ca
2
+
-binding domains of CPK4 in the degradation of CPK4.
In summary, the present study reveals a mechanism for the
rapid response of Arabidopsis to osmotic stress. The protein
levels of Ca
2+
-dependent protein kinase CPK4 exhibited sig-
nificance accumulation in response to hyperosmotic stress.
Moreover, PUB44, a U-box E3 ubiquitin ligase, ubiquitinated
CPK4 in ubiquitination reactions, both in vivo and in vitro.
Osmotic stress causes a reduction of CPK4 ubiquitination,
thus providing evidence that this contributes to the rapid
upregulation of CPK4 protein levels under osmotic stress
conditions.
Methods
Plant material and growth conditions
All plasmids used in this study were generated by Uracil-
Specific Excision Reagent (USER) cloning methods (Jorgensen
et al. 2017). All of the transgenic lines and mutants are in
the Arabidopsis (Arabidopsis thaliana) Col-0 ecotype. CPK4
OE PUB44 OE and CPK4 OE pub44 were generated by crossing
ProUBQ:mCherry-CPK4-myc line (Li et al. 2018) with ProUBQ:
Venus-PUB44-Flag and pub44, respectively. The Arabidopsis
plants were grown in growth room with conditions of 21 °C,
16/8 h light/dark cycle, 60% relative humidity,
120 μmol·m
−2
·s
−1
light (PRANDT, MPC-1100D-Light Emitting
Diode (LED)). Because of the temperature-sensitivity of
pub44 mutant, pub44 plants were grown in a growth chamber
at 25 °C under 16/8 h, 60% relative humidity, 120 μmol·m
−2
·s
−1
light (PRANDT, MPC-1100D-LED) to bulk up seeds.
For seedling growth analysis, after surface sterilization of
the seeds, stratification was conducted in the dark at 4 °C
for 3 days. Forty-five seeds of each genotype were sowed
on 1/2 MS plates (9 seeds of each genotype per plate) supple-
mented with 300 mM mannitol. At 6 days, the plates were
scanned, and primary root lengths were measured with
ImageJ software. For root elongation assays, seeds were strati-
fied for 3 day in the dark at 4 °C, sown, and grown on verti-
cally oriented 1/2 MS plates for 4 days. The seedlings with
similar primary root lengths were transferred onto new 1/2
MS plates lacking or supplemented with the 300 mM manni-
tol. The root tip position was labeled each day after transfer-
ring. After 6 days of growth, the plates were scanned, and
primary root lengths were measured with ImageJ software.
Yeast-two-hybrid assays
Yeast two-hybrid assays were performed with the
USER-modified pGBT9 and pGADGH vectors (Clontech)
(Jorgensen et al. 2017). Indicated bait and prey constructs
were transformed into yeast (Saccharomyces cerevisiae)
PJ69-4ɑ cells and selected on SD-L-W medium. Yeast colonies
were restreaked onto new SD-L-W plates and incubated for 1
or 2 days. Positive transformed clones were incubated in
SD-L-W liquid medium overnight, and then the OD600 of
cultures was adjusted to 1 with sterile water. A series of
2 μl 10-fold dilutions of transformants were spotted on
SD-L-W and SD-L-W-H supplemented with 2.5 mM
3-amino-1,2,4-triazole (3-AT) and grown for 3 days.
BiFC and subcellular localization analysis
For the BiFC assays, the full-length CPK4 was fused with
N-terminal Yellow Fluorescent Protein (YFP), and full-length
PUB44, PUB44 22-101, PUB44 102-699, and PUB44 700-801 were
fused with C-terminal YFP by USER cloning (Jorgensen et al.
2017). For transient expression in Nicotiana benthamiana leaves,
the indicated constructs were transformed into the
Agrobacterium (Agrobacterium tumefaciens) strain GV3101.
Overnight cultures of Agrobacterium were collected by centrifu-
gation at 4000 × g for 10 min. The pellets were washed twice
with 1 mL buffer (10 mM MES, pH5.6, 10 mM MgCl
2
, and
100 μM acetosyringone) and resuspended to OD600 = 1. Equal
volumes of bacterial suspensions were mixed and then injected
into the Nicotiana benthamiana leaves. Plants grew in the dark
for 1 day and then were transferred to long-day conditions for
2 days. For the observation of subcellular localization in
Arabidopsis, the transgenic lines expressing the ProUBQ:
Venus-CPK4-4myc and ProUBQ:Venus-PUB44-Flag were grown
PUB44 regulates CPK4 degradation THE PLANT CELL 2023: 35; 3870–3888 |3883
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on 1/2 MS medium for 5 days, and then treated with the 1/2 MS
liquid media with or without 100 mM NaCl for 8 h, the fluores-
cence signals in root tip cells were observed using Zeiss confocal
microscopy (LSM900, Laser Scanning Confocal Microscopy 900)
with parameters (Laser Wavelength 488 nm:5%, Emission
Wavelength 509 nm, Pinhole 5.00 AU/124 µm, Scan Speed 4,
Detector Gain 800).
Immunoprecipitation and in vitro pull-down assays
The IP of CPK4 and LC-MS/MS (Liquid Chromatography-
tandem Mass Spectrometry) analyses were performed as de-
scribed previously (Waadt et al. 2015). Pull-down assays were
performed as described previously (Wang et al. 2019). The
coding sequence of PUB44 was cloned into pGEX4T-1 to ob-
tain the GST-PUB44 construct. The coding sequence of
CPK4-myc was inserted into pET30a to generate a
His-CPK4-myc fusion protein construct. Fusion proteins
were expressed in Escherichia coli Rosetta strain with the in-
duction conditions (incubated with 0.5 mM isopropyl
β-D-1-thiogalactopyranoside (IPTG) overnight at 18 °C for
His-CPK4-myc or 0.5 mM IPTG overnight at 24 °C for
GST-PUB44). GST and His fusion proteins were purified with
Glutathione agarose (Yeasen) and HisSep Ni-Nitriloacetic
Acid Agarose Resin (Yeasen), respectively. Approximate
0.5 µg GST, GST-PUB44, and His-CPK4-myc protein and
10 µl agarose beads were incubated together in 1×
Phosphate Buffered Saline (PBS) buffer at 4 °C for 1 h. After
the beads were washed 8 to 10 times with wash buffer (1 ×
PBS, 0.1% Triton X-100 (v/v)), the bound proteins were eluted
by 1× SDS loading buffer, separated by 10% SDS-PAGE,
and subjected to immune-blot analysis using anti-GST
(SAB5300159, Sigma, 1:5000 dilution) or anti-myc antibody
(SAB1305535-40TST, Sigma, 1:5000 dilution).
Co-IP assay
Total protein from 12-day-old seedlings co-expressing
ProUBQ:mCherry-CPK4-myc and ProUBQ:Venus-PUB44-Flag
or ProUBQ:mCherry-CPK4-myc and ProUBQ:PUB25-Flag
were extracted with protein extraction buffer (50 mM Tris–
HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% gly-
cerol, 50 mM MG132, and 1× protease inhibitor mix).
Protein concentration was determined by Bicinchoninic
Acid kit (SK1060-1, Coolaber). An equal amount of total pro-
tein was mixed and incubated with flag-magnetic beads
(Thermo Fisher) at 4 °C overnight. The magnetic beads
were washed 4 times with the 1 × extraction buffer and
boiled with 1 × SDS loading buffer at 95 °C for 5 min. The
proteins were separated on SDS-PAGE and detected with
anti-myc (SAB1305535-40TST, Sigma, 1:5000 dilution) and
anti-Flag antibodies (F7425, Sigma, 1:5000 dilution). For
NaCl treatment, seedlings were incubated in 1/2 MS solution
with 150 mM NaCl for 4 h.
In vitro phosphorylation assays
In vitro phosphorylation, assays were performed as described
previously (Li et al. 2018). Recombinant CPK4, PUB44, and
RopGEF1 (Induction condition: 0.5 mM IPTG at RT over-
night) proteins prepared from E. coli were mixed in phos-
phorylation buffer (50 mM Tris–HCl pH 7.5, 10 mM MgCl
2
,
2 μM free Ca
2+
buffered by 1 mM EGTA, and CaCl
2
, 0.1%
Triton X-100, and 1 mM DTT). The in vitro phosphorylation
reactions were started by the addition of 200 μM ATP and 0.1
μCi·μL
−1
[γ-
32
P]ATP (PerkinElmer). The reactions were termi-
nated by the addition of SDS/PAGE sample buffer after
30 min incubation at room temperature. Proteins were sepa-
rated on SDS/PAGE, and the radioactivity of incorporated
32
P
in phosphorylated proteins was detected using an FLA-5000
PhosphorImager (Fujifilm). The protein level was analyzed by
Coomassie Brilliant Blue staining.
In vitro ubiquitination assay
In vitro ubiquitination assays were performed as described previ-
ously (Zhou et al. 2014). Recombinant proteins His-AtUBA1,
His-AtUBC8, His-CPK4-myc, and GST-PUB44 were purified
from Escherichia coli. 2 µg His-AtUBA1, 2 µg His-AtUBC8, 2 µg
His-CPK4-myc, 2 µg GST-PUB44, and 1 µg Flag-Ub (Boston
Biochem) were incubated in 60 µL of a 1× ubiquitination reac-
tion buffer (20 mM Tris–HCl pH 7.5, 5 mM MgCl
2
, 2 mM ATP,
0.5 mM DTT) at 30 °C for 2 h. The His-CPK4-myc protein was
immunoprecipitated from ubiquitination reaction mixtures
using the myc magnetic beads. After 2-h incubation at 4 °C,
the beads were washed 3 times with washing buffer (50 mM
Tris–HCl pH 7.5, 150 mM NaCl). The immunoprecipitates
were eluted using 1× SDS loading buffer. Samples were separated
on SDS-PAGE and detected using anti-myc (SAB1305535-40TST,
Sigma, 1:5000 dilution) or anti-Flag antibody (F7425, Sigma,
1:5000 dilution). Self-ubiquitination of GST-PUB44 was detected
with an anti-flag antibody (F7425, Sigma, 1:5000 dilution).
Bacterial ubiquitination system
The bacterial ubiquitination system was performed as
previously described (Han et al. 2017). The pCDFDuet-MBP-
CPK4-UBA1-S, pACYCDuet-PUB44-MYC-UBC8-S, and
pET28a-FLAG-Ub plasmids were sequentially transformed
into Escherichia coli BL21 cells. The bacteria were cultured
at 37 °C in 3 mL LB medium until OD
600
= 0.6. Then recom-
binant proteins were induced under the condition (IPTG
0.5 mM, 28 °C for 12 h). Bacteria were harvested by centrifu-
ging at 12,000 × g for 10 min, resuspended in 1× SDS loading
buffer and boiled at 95 °C for 5 min. Then crude protein ex-
tracts were separated by SDS-PAGE gels. Protein ubiquitina-
tion was detected with anti-myc (SAB1305535-40TST, Sigma,
1:5000 dilution) and anti-MBP antibodies (PA9060, Abmart,
1:4,000 dilution).
In vivo ubiquitination assays
Twelve-day-old ProUBQ:mCherry-CPK4-myc Col, ProUBQ:
mCherry-CPK4-myc pub44, ProUBQ:mCherry-CPK4-myc
ProUBQ:Venus-PUB44-Flag plants grown on 1/2 MS medium
were treated with 50 mM MG132 for 4 h and ground to pow-
der in liquid nitrogen. Total proteins were extracted with
protein extraction buffer (50 mM Tris–HCl pH 7.5, 150 mM
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NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol, 50 mM MG132,
and 1× protease inhibitor mix). The supernatants were cen-
trifuged twice at 12,000 × g for 15 min and passed through
0.45 µm filter. The resulting supernatants were incubated
with prewashed myc magnetic beads (Thermo Fisher) at 4
°C for overnight. The magnetic beads were washed 4 times
with the 1 × extraction buffer and boiled with 1 × SDS
loading buffer at 95 °C for 5 min. The proteins were
separated on SDS-PAGE and detected with anti-myc
(SAB1305535-40TST, Sigma, 1:5,000 dilution) and anti-Ub
rabbit polyclonal antibody (10201-2-AP, Proteintech,
1:1,000 dilution).
GUS staining
2 kb DNA sequence containing the promoter of PUB44 was
cloned into the modified pCAMBIA1301 vector by USER reac-
tion to drive GUS gene expression (Jorgensen et al. 2017). The
resulting construct was confirmed by sequencing and trans-
formed into Agrobacterium GV3101 strain. Homozygous T3
generation of Arabidopsis transgenic seedlings with single
T-DNA insertion were used for GUS staining. The primers
used for ProPUB44:GUS construction are listed in
Supplemental Table S1. GUS staining assays were performed
using a GUS staining kit (Coolaber).
Diaminobenzidine staining
Diaminobenzidine (DAB) staining to detect H
2
O
2
in situ was
conducted as described previously (He et al. 2012).
Five-day-old seedlings were treated with 300 mM mannitol
for 4 h, and then incubated in 0.1 mg/mL DAB (Sigma) dis-
solved in 50 mM Tris–Acetate (pH 5.0) for 8 h in the dark-
ness. The roots were rapidly washed 3 times with water
and observed under a microscope (OLYMPUS, BX53-LED).
Staining intensity was measured using Image J software.
Proline content measurement
12-day-old seedlings were transferred from 1/2 MS medium
into 1/2 MS liquid medium with or without 300 mM manni-
tol for 8 h. Roots were cut and ground in liquid nitrogen.
Proline concentration was measured using the proline deter-
mination kit (Solarbio).
Firefly luciferase complementation imaging (LCI)
assay
The full-length coding sequences of CPK4 and CPK12 ampli-
fied from Arabidopsis cDNA by PCR were cloned into the
pCAMBIA1300-LUCc vector to generate cLUC-CPK4 and
cLUC-CPK12. The full-length coding sequences of PUB44
were cloned into the pCAMBIA1300-LUCn vector to generate
the PUB44-nLUC construct. PUB44-nLUC and cLUC-CPK4
were co-transformed in Nicotianabenthamiana leaves. After
infiltration 48 h, a D-luciferin (Yeasen) solution was then in-
filtrated into the leaves, and luciferase activity was measured
using Promega-GloMax 20/20 Luminometer.
RNA extraction and reverse
transcription-quantitative PCR (RT-qPCR) analysis
Total RNA was isolated from 10-day seedlings using the RNA
Easy Fast kit (TianGen) according to the manufacturer’s re-
commendations. Reverse transcription was performed using
ProtoScript II First Strand cDNA Synthesis Kit (New England
Biolabs) from 5 µg of total RNA. RT-qPCR analysis was carried
out with Hieff qPCR SYBR Green Master Mix (Yeasen) on a
CFX96 machine (Bio-Rad). Primers used for RT-qPCR are
listed in Supplemental Table S1. The expression of ACTIN2
was used as an internal control.
Quantification and statistical analysis
The protein quantification was performed with ImageJ soft-
ware. Statistical analysis was performed with GraphPad
Prism. A two-tailed t-test was used to obtain P values and
the asterisks above bars represent the significant difference.
(Supplemental Data Set 1).
Accession numbers
The Arabidopsis Genome Initiative numbers for the genes
mentioned in this article are as follows: CPK4 (AT4G09570),
PUB44 (AT1G20780), CPK12 (AT5G23580), UBA1
(AT2G30110), UBC8 (AT5G41700), UBQ10 (AT4G05320), and
ACTIN2 (AT3G18780).
Acknowledgments
We are grateful to Dr. Libo Shan at Texas A&M University for
providing AtUBA1 and AtUBC8 constructs, and Dr.
Liangsheng Wang at China Agricultural University for critical
reading of the manuscript. We thank members of the Joint
Center for Single Cell Biology of Shanghai Jiao Tong
University (https://jcscb.sjtu.edu.cn/) for stimulating
discussions.
Author contributions
Z.L. and J.I.S. designed the research. W.F., X.L., Y.T., and X.W.
performed the experiments. Z.L. analyzed the data and wrote
the paper.
Supplemental data
The following materials are available in the online version of
this article.
Supplemental Figure S1. Immunoblot analyses of CPK4
protein abundance and confocal microscope observation of
Venus-CPK4 fluorescence under the indicated treatments.
Supplemental Figure S2. Identification of mutant and
transgenic plants.
Supplemental Figure S3. Identification of associated pro-
tein of CPK4 through IP-MS.
Supplemental Figure S4. Analysis of CPK4 phosphoryl-
ation, ubiquitination, and polymer formation.
PUB44 regulates CPK4 degradation THE PLANT CELL 2023: 35; 3870–3888 |3885
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Supplemental Figure S5. Hyperosmotic stress decreases
CPK4 and PUB44 ubiquitination levels.
Supplemental Figure S6. The pub44 mutant and
Venus-PUB44-Flag overexpression plants phenotype in nor-
mal conditions.
Supplemental Figure S7. PUB44 gene expression under NaCl
and mannitol conditions and PUB44 subcellular localization.
Supplemental Figure S8. CPK4 and PUB44 gene expres-
sion patterns derived from the eFP Browser database.
Supplemental Figure S9. Protein levels of various CPK4
variants with CHX and osmotic stress treatment.
Supplemental Table S1. Primer list.
Supplemental Data Set 1. Quantification and statistical
analysis.
Funding
This research was supported by grants from the National
Natural Science Foundation of China (NSFC31970297 to Z.L.),
a startup fund to Z.L. from Shanghai Jiao Tong University
and initial research and findings were funded by a grant from
the National Institutes of Health (GM060396) to J.I.S.
Conflict of interest statement. The authors declare no conflict of
interests.
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