Molecular Plant

Research Article

Phosphorylation of WHIRLY1 by CIPK14 Shifts

Its Localization and Dual Functions in Arabidopsis
Yujun Ren1,2, Yanyun Li1,2, Youqiao Jiang1, Binghua Wu1 and Ying Miao1,*
1
Center for Molecular Cell and Systems Biology, Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, College of Life Sciences, Fujian
Agriculture and Forestry University, Fuzhou 350002, China
2These authors contributed equally to this article.
*Correspondence: Ying Miao (ymiao2013@hotmail.com)
http://dx.doi.org/10.1016/j.molp.2017.03.011

ABSTRACT
Plastid-to-nucleus retrograde signaling is critical for normal growth and development in plants. The dual-
function and dual-located ssDNA binding protein WHIRLY1 (WHY1) has been proposed to coordinate the
retrograde signaling from plastids to the nucleus. However, the regulatory mechanism governing the func-
tional switch of WHY1 for mediating plastid-to-nucleus retrograde signaling remains unknown. Here, we
report that the Calcineurin B-Like-Interacting Protein Kinase14 (CIPK14) interacts with and phosphorylates
WHY1 in Arabidopsis. Phosphorylation of WHY1 results in increased accumulation in the nucleus and
enhanced binding with the promoter of WRKY53, which encodes a key transcription factor regulating
leaf senescence in Arabidopsis. Transgenic plants overexpressing CIPK14 showed an increased nuclear
isoform but decreased plastid isoform of WHY1, among which 95% of transgenic lines showed the stay-
green phenotype and 5% of lines showed the variegated pale-green phenotype. Interestingly, the pheno-
types of both types of transgenic plants could be recovered by overexpression of plastid-form WHY1. In
contrast, knockdown of CIPK14 caused early senescence and even seedling-lethal phenotypes along
with elevated expression of senescence-related genes such as WRKY53, SAG12, and NDHF but decreased
expression of MER11, RAD50, and POR genes, which could be rescued by overexpression of CIPK14 but not
by overexpressing plastid-form or nuclear-form WHY1; the stay-green plants overexpressing CIPK14
showed reduced expression of WRKY53, SAG12, NDHF, and large plastid rRNA. Consistently, the accu-
mulation of nuclear-form WHY1 was significantly reduced in the CIPK14 knockdown lines, resulting in
a low ratio of nuclear-/plastid-form WHY1. Taken together, our results demonstrate that CIPK14 regu-
lates the phosphorylation and organellar distributions of WHY1 and pinpoint that CIPK14 may function
as a cellular switch between leaf senescence and plastid development for coordinating the intercellular
signaling in Arabidopsis.
Key words: WHIRLY1, CIPK14, Retrograde Signalling, Leaf Senescence, Plastid Development
Ren Y., Li Y., Jiang Y., Wu B., and Miao Y. (2017). Phosphorylation of WHIRLY1 by CIPK14 Shifts Its Localization
and Dual Functions in Arabidopsis. Mol. Plant. 10, 749–763.

INTRODUCTION Leister, 2016). In addition, transcription factors that are able

Intercellular communication is important for the lifespan of a
cell and may become altered with age. Outside the nucleus,
the two DNA-containing organelles, plastid and mitochondrion,
utilize specific signaling molecules to convey information of
their developmental and physiological states into the nucleus
and thereby modulate the expression of nuclear genes. This
retrograde signaling is critical for proper plant development.
Many compounds, such as reactive oxygen species, chloro-
phyll biosynthesis metabolites, and redox system components,
as well as plastid-derived small metabolites have been sug-
gested as retrograde signals (Kleine et al., 2009; Kleine and
to relocate from the chloroplast to the nucleus (Sun et al.,
2011; Isemer et al., 2012; Avendano-Vazquez et al., 2014), or
those that could be released from the endoplasmic reticulum
(ER) upon proteolytic activation in mitochondrial retrograde
regulation (DeClercq et al., 2013; Ng et al., 2013a, 2013b),
as well as the classical inducible nuclear transcription factors
(Koussevitzky et al., 2007; Van Aken et al., 2013), are also
considered to be involved in retrograde signaling. However,
Published by the Molecular Plant Shanghai Editorial Office in association with
Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

Molecular Plant 10, 749–763, May 2017 ª The Author 2017. 749
Molecular Plant Phosphorylation of WHY1 by CIPK14 Affects Its Dual Functions
all these transcription factors seem to play indirect roles in
regulating gene expression or the maintenance of plastid DNA.
The mechanism underlying a direct contribution of proteins in
transferring information from plastids or mitochondria to the
nucleus has not yet been envisaged.
Members of the WHIRLY family, which have a tetrameric structure
resembling whirligigs (Desveaux et al., 2002), are dual-located
proteins both in the nucleus and organelles and perform
numerous cellular functions (Krause et al., 2005; Grabowski
et al., 2008). They were first discovered as transcriptional
activators binding to the elicitor response element in the
promoter region of pathogenesis-related (PR) genes in potato
(Solanum tuberosum) and Arabidopsis (Desveaux et al., 2000,
2004). In Arabidopsis, they were able to bind various DNA
sequences such as the telomeres (Yoo et al., 2007), the distal
element upstream of a kinesin gene (Xiong et al., 2009), the
promoter of the early senescence marker gene WRKY53 in a
development-dependent manner (Miao et al., 2013), and the
promoter of the senescence-associated gene HvS40 in barley
(Hordeum vulgare) (Krupinska et al., 2014). In maize (Zea mays)
chloroplasts, WHIRLY1 (WHY1) was proposed to bind both
DNA and RNA and play a role in intron splicing (Prikryl et al.,
2008), while in barley (Hordeum vulgare) chloroplasts, WHY1
was found to be associated with intron-containing RNA process-
ing (Melonek et al., 2010). In addition, it was also demonstrated
that WHIRLY proteins in organelles could function as an anti-
recombinant protein favoring accurate DNA repair for main-
taining organelle genome stability (Cappadocia et al., 2010,
2012; Lepage et al., 2013). All these studies suggest that
WHIRLY proteins might function distinctly depending on their
intracellular localization and/or the different developmental
stages of plants.
There were three WHIRLY members (WHY1, WHY2 and WHY3) in
Arabidopsis but only two members (WHY1 and WHY2) in mono-
cot plants. A recent study showed that a why1why3polIb-1
mutant defective in WHY1, WHY3, and the DNA polymerase 1B
(Pol1B), a type I chloroplast DNA polymerase, exhibited a severe
yellow-variegated phenotype (Lepage et al., 2013). Compared
with the wild-type, this mutant showed lower photosynthetic
electron transport efficiencies and had higher accumulation
of reactive oxygen species and altered expression of a number
of oxidation-related nuclear genes (Lepage et al., 2013).
Based on these observations, it was proposed that WHY
proteins may mediate a link between the chloroplast to
nucleus signaling for enhanced adaptation to oxidative
stress (Lepage et al., 2013). In barley, WHY1 influenced the
expression of a specific subset of genes encoding chloroplast
proteins; severely reduced expression of WHY1 resulted in
reduced sensitivity of photosynthesis to low nitrogen, owing
to a possible disruption of retrograde signaling between
the plastid and the nucleus (Comadira et al., 2015). Moreover,
the close association of WHY1 with components of the
thylakoid electron chain and other evidence (Krause and
Krupinska, 2009; Isemer et al., 2012) led Foyer et al. (2014)
to propose that WHY1 is a photosynthetic redox sensor for
chloroplast to nucleus retrograde signaling, and the
movement of WHY1 from the chloroplasts to the nucleus
could be triggered by the redox state of the photosynthetic
electron transport chain. Yet the question remains how WHY1
750 Molecular Plant 10, 749–763, May 2017 ª The Author 2017.
is sequestered to enter the nucleus and to switch its functions
at a certain stage of plant development or after modification
by other factors.
In the present study, we identified CIPK14 as a WHY1 interacting
partner. This SNF1-related protein kinase was able to phosphor-
ylate WHY1 protein in the nucleus. Phosphorylation of WHY1
by CIPK14 regulated its subcellular localization and distribution
ratio between plastids and the nucleus, thereby switching its
function from controlling ribosome RNA stability in plastids to
senescence retardation in the nucleus.
RESULTS
Intracellular Accumulation of WHY1 Is Developmentally
Regulated during Leaf Aging
To examine the subcellular accumulation abundance of WHY1,
we used transgenic plants expressing a fusion of WHY1 with
the HA (human influenza hemagglutinin) epitope tag under the
control of its own promoter (PWHY1-HA; Miao et al., 2013).
Leaves from 6-week-old transgenic plants were used for the
preparation of chloroplast fractions (membranes and stroma)
and a nuclear fraction. Western blotting with anti-HA antibody
revealed that the monomeric form of WHY1 was enriched in
the stroma fraction and nucleus. An additional protein band
with a slightly higher molecular weight was detectable in the
nucleus (Figure 1A). This protein band disappeared when
treated with phosphatase (Figure 1B), suggesting that it might
represent a phosphorylated form of WHY1. To further monitor
the phosphorylation of WHY1 during leaf development, we
isolated plastid and nuclear proteins from 3- to 8-week-old
rosette leaves of PWHY1-HA plants, separated them on high-
Tris SDS–PAGE, and analyzed with antibody against the HA
tag. The result showed that WHY1 accumulation in plastids grad-
ually declined from the 3-week-old stage, whereas nuclear WHY1
protein showed a dephosphorylated form in the 3-week-old
leaves, both the dephosphorylated and phosphorylation forms
in the 4- to 6-week-old leaves, and a predominantly phos-
phorylated form in the 6- to 8-week-old leaves (Figure 1C).
The phosphorylated and dephosphorylated forms of nuclear
WHY1 protein were confirmed upon phosphatase treatment.
Moreover, the amount of nuclear WHY1 protein gradually
increased upon phosphatase treatment (Figure 1C). These
results indicate that the phosphorylation status of WHY1 is
developmentally regulated.
CIPK14 Interacts with WHY1 in the Nucleus
To identify the kinase(s) responsible for phosphorylation of
WHY1, we prepared a cDNA expression library from leaves of
5- and 7-week-old plants, when WHY1 achieved its first expres-
sion peak during plant development (Miao et al., 2013), and
performed a yeast two-hybrid screen to look for its interacting
partners. We used truncated WHY1, in which the activation
domain (i.e., the last 33 amino acids at the C terminus) is deleted
(Pfalz et al., 2005), fused to downstream of the plastid transit
peptide (32 amino acids) and the c-myc tag, as the bait. More
than 30 positive colonies were obtained and sequenced.
Among them, the full-length cDNAs of 11 positive colonies were
isolated and confirmed for their interactions with WHY1 by fresh
re-transformation, respectively (Supplemental Figure 1). One of
Phosphorylation of WHY1 by CIPK14 Affects Its Dual Functions
Figure 1. WHY1 Protein Immunodetection in Different
Compartments of Arabidopsis Cells from the Pwhy1-WHY1-HA
Line and at Different Development Stages.
(A) WHY1 protein isolated from plastid and the nucleus fractions of
6-week-old rosette leaves.
(B) Nuclear WHY1 and nuclear WHY1 treated with phosphatase from
6-week-old rosette leaves. Asterisk indicates the phosphorylated form
of WHY1.
(C) 12 mg of plastid WHY1 and nuclear WHY1 or nuclear WHY1 treated
with phosphatase isolated from 3- to 8-week-old Arabidopsis rosette
leaves.
An antibody directed against the HA tag was used for detection of the
WHY1 fusion proteins, The purity of the subcellular protein fractions
was shown by immunological detection of the plastid protein cyto-
chrome b559 (a-Cytb559) and the nuclear protein histone H2B (a-His-
tone H2B). Asterisk indicates the phosphorylated form of WHY1.
the candidate proteins interacting with WHY1 was identified
as CIPK14, a CBL-interacting serine/threonine-protein kinase
(in the family of SNF1-related kinases; see Luan, 2009). The
CIPK14-GFP fusion protein was localized both in the cytoplasm
and the nucleus of onion epidemic cells and Arabidopsis proto-
plasts (Figure 2A-a,f).
To confirm that CIPK14 and WHY interact in vivo, we performed a
bimolecular fluorescence complementation assay. Cotransfor-
mation of the protoplasts with the constructs CIPK14-cmyc-
GFPn173 and WHY1-HA-GFPc155 containing the full-length
WHY1 resulted in fluorescent signals both in the nucleus and
cytoplasm. However, the fluorescent signal in the cytoplasm dis-
appeared 4 h after transformation (Figure 2A-g). In contrast,
cotransformation of CIPK14-cmyc-GFPn173 and nWHY1-HA-
GFPc155 that expresses the WHY1 without plastid transit
peptide generated fluorescent signals only in the nucleus
(Figure 2A-i). Consistent with previous results (Grabowski et al.,
2008), expression of the full-length WHY1 fused with GFP
showed fluorescence in plastids (Figure 2A-d), but expression
of nWHY1 fused with GFP generated fluorescence in the
nucleus (Figure 2A-e). We confirmed the protein expression of
Molecular Plant
both CIPK14 and WHY1 by western blot analyses using GFP-
specific or HA-specific antibody (Figure 2B). From the above
results, it seems that overexpression of CIPK14 enhanced
WHY1 protein accumulation in the nucleus.
To further investigate whether WHY1 and CIPK14 interact
only in the nucleus, we conducted a co-immunoprecipitation
assay using nuclear proteins and plastid proteins isolated from
5-week-old rosette leaves of double transgenic plants (PWHY1-
HA plus oeCIPK14-GFP). The fraction pulled down by the anti-
body against HA tag was analyzed for the presence of CIPK14-
GFP using an antibody against GFP. The CIPK14 protein (about
70 kDa) was only detected in nuclear proteins (and nuclear
plus cytoplasm proteins), but not in plastid proteins (Figure 2C),
suggesting that the interaction between WHY1 and CIPK14
occurred predominantly in the nucleus.
Phosphorylation of WHY1 by CIPK14 Enhances Its
Binding with the Promoter of WRKY53
Since CIPK14 is a kinase, the interaction between CIPK14 and
WHY1 prompted us to test whether CIPK14 could phosphorylate
WHY1. An in-gel kinase assay using recombinant WHY1 and
CIPK14 proteins showed a strong band of 28 kDa correspond-
ing to phosphorylated WHY1 (Figure 3A). In contrast, a mutated
version of CIPK14, CIPK14T168D, in which the Thr168 located at
the kinase activation loop (Guo et al., 2001) was replaced by
Asp, was not able to phosphorylate WHY1 in vitro (Figure 3A).
Phosphorylation of WHY1 by CIPK14 was further confirmed
using cell extracts from 6-week-old transgenic plants overex-
pressing CIPK14 (oeCIPK14) and the wild-type plants, whereas
phosphorylation of WHY1 was not detected from the kdCIPK14
plants, in which the expression of CIPK14 was knocked down
(Figure 3B).
To examine WHY1 phosphorylation status in vivo under various
CIPK14 expression levels, the nuclear extracts from 5-week-old
rosette leaves of PWHY1-HA plants and PWHY1-HA plants
overexpressing CIPK14 (oeCIPK14/PWHY1-HA) or knockdown
CIPK14 (kdCIPK14/PWHY1-HA) were isolated and subjected to
immunodetection using high-Tris PAGE gel; phosphorylated
bands of WHY1-HA were detected in the oeCIPK14/PWHY1-
HA plants, while dephosphorylated bands of WHY1-HA accumu-
lated more strongly in the kdCIPK14/PWHY1-HA plants by both
HA antibody and the Thr168 position phosphor antibody. In the
plastid extracts from these transgenic plants, the protein amount
of WHY1-HA slightly declined in the oeCIPK14/PWHY1-HA
plants but significantly increased in the kdCIPK14/PWHY1-HA
plants, nevertheless showing no phosphorylation phenomenon.
All these results indicate that CIPK14-mediated phosphorylation
of WHY1 only occurred in the nucleus and was correlated to
CIPK14 expression, whereas the plastid form of WHY1 strikingly
showed no detectable phosphorylation (Figure 3C) but with
changed protein amount.
As previously reported, WHY1 was able to bind to the pro-
moter of WRKY53 (Miao et al., 2013). We then examined the
effect of CIPK14-mediated WHY1 phosphorylation on its
DNA-binding affinity to WRKY53 promoter by ChIP–PCR
and electrophoretic mobility shift assay (EMSA). The chro-
matins were prepared from the 5-week-old rosette leaves of
Molecular Plant 10, 749–763, May 2017 ª The Author 2017. 751
Molecular Plant Phosphorylation of WHY1 by CIPK14 Affects Its Dual Functions

Figure 2. Localization of CIPK14 Protein and Interaction of CIPK14 and WHY1 by Bimolecular Fluorescence Complementation Assay
and CoIP.
(A) Onion epidermal cells or Arabidopsis wild-type protoplasts were transformed with 35S:CIPK14-GFP. Green fluorescence can be detected in the
cytoplasm and the nucleus (a, f). Onion epidermal cells were transformed with 35S:WHY1-GFP and 35S:nWHY1-HA-GFPc155 plus 35S:nWHY1-cmyc-
GFPn173 as positive controls (d, e) (Grabowski et al., 2008). Onion epidermal cells and Arabidopsis protoplasts were transformed with 35S:WHY1-HA-
GFPc155 plus 35S:CIPK14-cmyc-GFPn173 and 35S:nWHY1-HA-GFPc155 plus 35S:CIPK14-cmyc-GFPn173. Green fluorescence can be detected (left)
in the cytoplasm and the nucleus according to the time course (2, 4, 10, and 16 h) and bright field (right) (b, c, g, and i). WHY2 protein instead of WHY1 was
used as a negative control (h). The bar represents 100 mm.
(B) Western blot detection of CIPK14-GFP and WHY1-HA proteins isolated from the transformed protoplast according to the time course (2, 4, 10, and 16
h) using antibodies against GFP peptide and HA tag.
(C) The interaction detection of CIPK14 and WHY1 in the nucleus by co-immunoprecipitation. Protein extracts (plastid protein, P; nuclear protein, N; and
nuclear plus cytoplasm protein, N + C) of overexpressing CIPK14-GFP in a PWHY1-HA background line as input. CoIP with antibody against an HA tag;
the antibody against GFP was used to immunodetect the pull-down protein.

oeCIPK14/ID-nWHY1-HA, kdCIPK14/ID:nWHY1-HA, and ID-
nWHY1-HA transgenic lines (see Methods section) after 4 h
induction by 20 mM estradiol treatment; the induced nWHY1
were confirmed by western blot (Figure 3D-b). The ChIP–qPCR
experiment with anti-HA antibody showed that Pwrky53 and
Pwrky33 element enrichment was more than 2-fold higher in the
oeCIPK14/ ID-nWHY1-HA, but largely decreased in the
kdCIPK14/ID-nWHY1-HA compared with the ID-nWHY1-HA
plants (Figure 3D-a), Actin was used as an inner control. A
series of EMSA experiments using purified recombinant WHY1
752 Molecular Plant 10, 749–763, May 2017 ª The Author 2017.
protein in the presence of ATP and CIPK14 protein revealed that
the binding affinity of the WHY1 protein to the promoter
fragment of WRKY53 increased with incubation time (Figure 3E).
However, the mutated CIPK14 (T168D) did not show any effect
on the binding affinity with extended incubation time (Figure 3F),
indicating that the phosphorylation of WHY1 by CIPK14
enhanced DNA–protein complex formation. Moreover, when the
nuclear proteins, which were extracted from the 6-week-old
rosette leaves of the oeCIPK14/PWHY1-HA line, kdCIPK14/
PWHY1-HA line, or PWHY1-HA plants were incubated with
Phosphorylation of WHY1 by CIPK14 Affects Its Dual Functions Molecular Plant

Figure 3. The Phosphorylation of WHY1 by CIPK14 Affects the Binding Affinity of WHY1 at the WRKY53 Promoter by ChIP-qPCR and
EMSA.
(A and B) The phosphorylation of WHY1 by the recombinant purified CIPK14 kinase (A) and plant extracts (B) from oeCIPK141(oe5), kdCIPK14(kd1), and
wild-type plants (wt) in an in vitro gel kinase assay. CIPK14, purified recombinant protein expressed in E. coli; WHY1, purified recombinant protein
expressed in E. coli; CIPK14m, purified recombinant protein CIPK14 mutated 168T/D expressed in E. coli; CKII and casein used as controls.
(C) The phosphorylation status of nuclear and plastid WHY1 protein isolated from 6-week-old rosette leaves of oeCIPK14 (oe5:oeCIPK14-5;oe6:oe-
CIPK14-6), kdCIPK14 (kd1:kdCIPK14-1;kd4:kdCIPK14-4) in a PWHY1-HA background and PWHY1-HA plants. Asterisk indicates the phosphorylated
form of WHY1. An antibody against Pi-Thr168 (provided by the Biochemistry Department of Tuebingen University) was used to immunodetect the
phosphorylated form WHY1. Coomassie staining shows the same amount of loading proteins.
(D) The enrichment of WRKY53 and WRKY33 promoter (Pwrky53, Pwrky33) of WHY1-targeted genes detected by ChIP–qPCR (a) using 5-week-old
rosette leaves of oeCIPK14 (oe5), kdCIPK14 (kd1) in the ID-nWHY1-HA background, and ID-nWHY1 plants in the wild-type background after 4 h induction
with 20 mM estradiol. The induced nWHY1-HA protein was detected by antibody against an HA tag (b). Asterisks (*P < 0.05, **P < 0.01, ***P < 0.001) show
significant differences from the ID-nWHY1 line according to Student’s t test. Actin was used as an inner control for ChIP–qPCR.
(E) The binding affinity of WHY1 to the Pwrky53 element after incubating with plant extract proteins from oeCIPK14 (oe5,oe6), kdCIPK14 (kd1,kd4), and
wild-type (wt) in a PWHY1-HA background. 25x, 50x, 25-fold and 50-fold specific competitor; Pwrky53 element, GTCAAAT.AAAAT (see Miao et al.,
2013). The western blot with HA tag antibody after incubation of the plant extract proteins shows the same amount of WHY1 proteins.
(F and G) The binding affinity of WHY1 to the Pwrky53 element after incubating with recombinant CIPK14 and CIPK14m protein from E. coli. With a time
course (0,1, 2, 3, 4, and 5 h), the Pwrky53 element was incubated with purified CIPK14(F) and CIPK14m(G). 25x, 50x, 25-fold, and 50-fold specific
competitor; Pwrky53 element, GTCAAAT.AAAAT (see Miao et al., 2013). The western blot with His tag antibody after incubation of the recombinant
WHY1 proteins shows the same amount of WHY1 proteins.

Pwrky53 element by EMSA, a stronger signal appeared for
the oeCIPK14/PWHY1-HA line and a much weaker signal for
the kdCIPK14/PWHY1-HA line, compared with the PWHY1-HA
plants (Figure 3G). The loading control in the gel shift experiment
was done using western blot with antibody against 6xHis tag
and HA tag. Both ChIP–qPCR in vivo and the EMSA DNA-
binding assay in vitro demonstrated that phosphorylation of
WHY1 by CIPK14 enhances the binding of WHY1 with the pro-
moter of WRKY53.
Alteration of CIPK14 Expression Level Produces
Multiple Phenotypes
Under illumination for16 h, seedlings of a CIPK14 T-DNA insertion
line (Supplemental Figure 2A) grew yellow from the old leaf to
young leaf and died 20 days after germination, showing
severe senescence and an early seedling-lethal phenotype
(Figure 4A). To revise this lethalness, a CIPK14 interference
knockdown line was generated (Supplemental Figure 2C). The
plants showed an early senescence and cell death phenotype
(Figure 4B and 4D). In addition, a CIPK14-overexpressing line
was produced (Supplemental Figure 2B), and the CIPK14
mRNA level of different knockdown or overexpression lines was
quantified by qRT–PCR (Supplemental Figure 2D). In the
oeCIPK14 lines, 5% of plants of a total 205 T1 transgenic lines
unexpectedly displayed variegation patterns with pale-green
plants (oeCIPK14var) and the other 95% of plants showed
a stay-green phenotype (Figure 4B). The fluorescence of
chlorophyll of oeCIPK14var leaves was visualized with a strong
diminution in chlorophyll autofluorescence compared with
Molecular Plant 10, 749–763, May 2017 ª The Author 2017. 753
Molecular Plant Phosphorylation of WHY1 by CIPK14 Affects Its Dual Functions
wild-type plants under confocal microscopy (Figure 4B). At the
end, two CIPK14-overexpressing lines (oeCIPK14-5 and
oeCIPK14-6) and two RNAi knockdown lines (kdCIPK14-1 and
kdCIPK14-4) (Supplemental Figure 2) were subjected to
phenotype profiling. Thirty individual plants of each line were
categorized by grouping the leaves at 10 weeks according to
their color (green; green/yellow; fully yellow; brown/dry;
Figure 4C and 4D). By this, two CIPK14-knockdown lines
showed less numbers of rosette leaves, early bolting at
4.5 weeks after germination, and an early yellowing phenotype at
week 7, then sharply got brown with cell death after week 9;
two CIPK14-overexpressing lines showed a delay senescent
phenotype compared with wild-type (Figure 4C and 4D). The
chlorophyll content was measured from the whole rosette of
10-week-old plants (Figure 4E). In the kdCIPK14 lines, it was
20%–30% of that of wild-type plants, whereas in the
oeCIPK14-5 and oeCIPK14-6 lines, it was 70%–180% of the
wild-type; however, the variegated pale-green plants contained
chlorophyll less than 40% of the wild-type (Figure 4E). This
variegated pale-green phenotype was rather similar to why1why3
(Maréchal et al., 2009), why1why3polIb-1 (Lepage et al., 2013),
and ZmWHY1 phenotype (Prikryl et al., 2008; Zhang et al., 2013).
CIPK14 Modulates the Intracellular Distribution of WHY1
between the Plastids and the Nucleus
Having shown that CIPK14 expression level changed the
phosphorylation status of WHY1 protein in the nucleus and the
754 Molecular Plant 10, 749–763, May 2017 ª The Author 2017.
Figure 4. Phenotype Analyses of CIPK14
Mutants.
(A) A T-DNA insertion line (a, koCIPK14 homo-
zygous line; b, koCIPK14 heterozygous line; c,
wild-type) of the CIPK14 gene shows a lethal
phenotype.
(B) 5% variegation pale-green phenotypes
(oevar:oeCIPK14var) shown in oeCIPK14 lines;
chlorophyll autofluorescence of the oevar line and
the wild-type (wt) are observed under confocal
microscopy.
(C) The phenotypes of the leaves from plants
grown for 10 weeks are observed by arrangement
of leaves according to their age with number
one being the oldest and a whole rosette upside
down. An early senescence phenotype in two
kdCIPK14 lines (kd1 and kd4) and a stay-green
phenotype in two oeCIPK14 lines (oe5 and oe6)
compared with wild-type are presented.
(D) Senescent leaf proportions are characterized
by 30 plants at week 10. The error bars indicate
the standard error of 2 3 6 independent mea-
surements.
(E) Chlorophyll content of CIPK14 mutants at
week 10; wt is set to 100%. The error bars
indicate the standard error of five independent
measurements.
allocation of its non-phosphorylated form
in plastid, in order to further demonstrate
that CIPK14 might function as a phosphory-
lation shift of WHY1 allocation between
plastids and the nucleus, we conducted
immunodetection on the ratio of nuclear and plastid WHY1 pro-
tein in 5-week old rosette leaves of oeCIPK141/PWHY1-HA or
kdCIPK14/PWHY1-HA mutant lines. Since the different CIPK14
mutant lines expressed variable amounts of CIPK14 proteins,
the ratio of plastid and nuclear isoforms of WHY1 changed
accordingly (Figure 5A and 5B). The amount of plastid isoform
WHY1 protein decreased in the oeCIPK14 line, but increased in
the kdCIPK14 line compared with the control plant PWHY1-HA.
The amount of nuclear isoform of WHY1 did not change signifi-
cantly, instead appeared more as phosphorylated proteins in
the oeCIPK141 lines. On the other hand, nearly all dephosphory-
lated protein was observed in the kdCIPK14 lines (Figures 3C
and 5A).
Since plastid-localized WHY1 had implied functions on RNA
processing, the knockout WHY1 gene showed a plastid ribo-
some defect phenotype in maize and in barley (Prikryl et al.,
2008; Melonek et al., 2010; Zhang et al., 2013), the altered
allocation of plastid WHY1 by CIPK14 expression prompted
us to test whether the above-mentioned 5% variegated pale-
green leaves in the oeCIPK14var line and the ‘‘stay-green’’
phenotype of the oeCIPK14 lines had a relevant mechanism
for RNA processing. To this end, total RNAs isolated from
oeCIPK14var and oeCIPK14 plants were subjected to RNA
gel separation, and dot plots were conducted with different
dilutions of RNA and northern blot analyses with probes for
16S, 23S, 4.5S, 5S rRNA, RPL, and RPS. The results showed
that in the oeCIPK14var line, whole-plastid ribosomal RNAs
Phosphorylation of WHY1 by CIPK14 Affects Its Dual Functions
(D) Northern blot analyses of chloroplast 4.5S, 5S, 16S, 23S, RPL, RPS, and cytoplasmic 18S rRNAs. Total RNA (3 mg) from 4-week-old leaves of CIPK14
mutants were used for the northern blot experiments. Equal loading controls for northern blot analyses are shown in 18S.
were reduced, suggesting a ribosome deficiency. The stay-
green plants (oeCIPK14-5 and oeCIPK14-6) exhibited a defect
in the biogenesis of the large ribosomal subunit (Figure 5C–
5D). The phenotypic similarity between the pale-green plants
and the ZmWHY1 knockout mutant as well as between the
stay-green plants and the ore4 mutant in which the levels of
RPS17 mRNA and 16S rRNA were reduced (Woo et al., 2002)
both implied a similar pWHY1 function involved in plastid
RNA processing.
To further confirm that the above phenotype of CIPK14
mutants was due to the alternative isoform WHY1, the con-
structs overexpressing plastid isoform WHY1 (oepWHY1-HA),
and nuclear isoform WHY1 (oenWHY1-HA) plants were
transformed to oeCIPK14-5, oeCIPK141var, and kdCIPK14-1
transgenic plants, respectively (Supplemental Figure 3).
The homozygous double transgenic plants of oepWHY1/
oeCIPK14-5 or oepWHY1/oeCIPK14var recovered their
background plant phenotype showing a wild-type-like
phenotype (Figure 6A–6B) with increased pWHY1 accumulation
in plastids (Figure 6C). However, transgenic plants of
oenWHY1/oeCIPK14-5 enhanced the stay-green phenotype of
oeCIPK14-5 lines, and those of oenWHY1/oeCIPK14var did not
significantly change the oeCIPK14var phenotype (Figure 6A–
6B), both of which showed increased amount of nWHY1 protein
in the nucleus (Figure 6C); neither pWHY1 nor nWHY1
ameliorated the yellowing phenotype of kdCIPK14, and even
enhanced yellowing (Figure 6A–6B and Supplemental
Figure 2E). To understand molecular basis underlying the
observed phenotypes, we examined the expression of
senescence-related target genes of WHY1. Only expression of
SAG12 was significantly upregulated in the oenWHY1/kdCIPK14,
while expression of NDHF, SAG101, and PDF1.2 were largely up-
regulated in the oepWHY1/kdCIPK14 compared with the
kdCIPK14 line. In contrast, WRKY53 expression showed little
change in both double lines compared with the kdCIPK14 line
Molecular Plant
Figure 5. WHY1 Protein and the Plastid
Ribosomal RNA Analyses of oeCIPK14
(oe5:oeCIPK14-5; oe6:oeCIPK14-6; var:
oeCIPK14var), kdCIPK14 (kd1:kdCIPK14-1;
kd4:kdCIPK14-4) in a PWHY1-HA Back-
ground and PWHY1-HA Plants.
(A and B) The distribution of plastid WHY1 pro-
tein and nuclear WHY1 protein from 5-week-
old rosette leaves of oeCIPK14, kdCIPK14 in a
PWHY1-HA background and PWHY1-HA plants
(A). Antibodies against an HA tag and Pi-Thr168
were used for immunodetection; Coomassie
staining was used as a loading control. The pro-
tein band signal was captured and calculated by
the ImageJ program (http://www.di.uq.edu.au/
sparqimagejblots) (B). The data are the average
of three replicates.
(C) Dot blots of total RNA (1 mg, 5 mg, 15 mg) iso-
lated from 4-week-old rosette leaves of CIPK14
mutants performed with plastid rDNA (4.5S, 5S,
16S, 23S rDNA), ribosome protein with large and
small subunits (RPL and RPS), ORF23 and ORF63
as probes, with pBluescript plasmid DNA as the
control.
(Figure 6D). These results further indicated that CIPK14
phosphorylates nWHY1 and determines the distribution ratio of
pWHY1 and nWHY1 proteins, which determines the variegation
pale-green or early senescence phenotype of transgenic plants
overexpressing CIPK14.
CIPK14 Regulates the Expression of WHY1 Target
Genes
To further define the CIPK14-WHY1 regulatory network, we
examined the expression of 30 putative targeted genes of
WHY1, some WHY1 function-related genes, as well as the
three WHIRLY family members in the CIPK14 mutants (Miao
et al., 2013; Comadira et al., 2015). As expected, changes
in the transcript levels of a number of senescence- and
cell-death-related genes such as WRKY53, SAG12, WRKY33,
SAG101, NDHF, PDF1.2-3, and double-strand-break (DSB)-re-
lated genes such as MER11 and RAD50 (Vannier et al., 2006;
Ronceret et al., 2009), as well as chlorophyll syntheses-
related enzyme protochlorophyllide oxidoreductase gene
(POR), were observed (Figure 7A and 7B); Overexpressing
or knocking down CIPK14 had no obvious effect on
gene expression of WHY1, WHY2, and WHY3, but both
significantly promoted PDF1.2a, PDF1.2b, PDF1.3, and
SAG101 transcript levels. Overexpression of CIPK14
significantly downregulated the expression of WRKY53,
WRKY33, SAG12, and NDHF genes as well as the levels
of plastid ribosomal RNAs except for 5S rRNA, whereas
the transcript levels of MER11, RAD50, and POR were
upregulated. Consistently, knockdown of CIPK14 upregulated
the expression of WRKY53, WRKY33, SAG12, and NDHF
(Zapata et al., 2005) but downregulated the expression of
MER11, RAD50, and POR (Figure 7A and 7B). These results
indicate that alteration of CIPK14 did not change the WHY1
transcript level but did modify the phosphorylation status of
nWHY1 and the distribution ratio of pWHY1 and nWHY1
Molecular Plant 10, 749–763, May 2017 ª The Author 2017. 755
Molecular Plant Phosphorylation of WHY1 by CIPK14 Affects Its Dual Functions
proteins, resulting in the remodeling of gene expression
patterns of downstream-targeted genes.
DISCUSSION
A fundamental question in plant biology is how information
exchanges between plastids and the nucleus coordinate
cellular function. In the work reported here, we have provided
evidence that a serine/threonine SNF1-related protein kinase
(CIPK14) was able to interact with the dual-targeted WHY1 pro-
tein, altering its phosphorylation status, and in this way provided
a switch controlling its localization, distribution, and function in
either plastids or the nucleus.
Plastid-to-nucleus retrograde signaling was well known for playing
roles in coordinating the proper expression of those nuclear genes
encoding plastid-localized proteins associated with plastid devel-
opment and nuclear events. However, the molecular connections
are still largely unknown. The dual-located protein WHY1 was pro-
posed as a retrograde signaling molecule between organelles and
the nucleus (Krause and Krupinska, 2009). There might be two
756 Molecular Plant 10, 749–763, May 2017 ª The Author 2017.
Figure 6. Genetic Complementation of
CIPK14 Mutants with Overexpression of
Plastid WHY1 and Nuclear WHY1 Partially
Recovers the CIPK14 Mutant Phenotype.
(A) Phenotype analyses of a series of double
mutants of CIPK14 and WHY1 (Supplemental
Figure 3) Leaves from the transgenic plants are
laid out in order of leaf emergence as indicated
by the numbers. Number 1 is the oldest. Results
from one of five biological replicates are shown.
(B) The proportion of senescent leaves is
characterized by 20 plants at week 7. The error
bars indicate the standard error of 2 3 6
independent measurements.
(C) 12 mg of plastid (P) and nuclear (N) protein
from 4-week-old transgenic plants were im-
munodetected by antibody against an HA tag.
WB, western blot.
(D) The gene expression of WHY1 downstream
senescence-related genes detected in a series of
complementation lines of CIPK14 and WHY1
mutants by qRT–PCR. The relative expression
level of the gene is normalized to Actin, with the
wild-type as 1. The standard error bars present
three time biological replicates and three tech-
nical replicates, n = 3 3 3 = 9; the values are
shown as means ± SD. Asterisks (*P < 0.05, **P <
0.01, ***P < 0.001) show significant differences
compared to the kdCIPK14 line according to
Student’s t test.
hypotheses: first, the WHY1 protein in
plastids produces or affects a retrograde
signal, for example, small metabolites,
reactive oxygen species (Lepage et al.,
2013; Comadira et al., 2015), or hormone
accumulation (unpublished results in our
lab). The why1why3polIb-1 mutants of
Arabidopsis accumulated a higher amount
of reactive oxygen species (Lepage et al.,
2013). Knockdown WHY1 in barley affected
the expression level of NDH as well as the expression of
photosystem-related genes, suggesting WHY1 might be a
potential target for redox regulation (Foyer et al., 2014; Comadira
et al., 2015) and link the chloroplast signal to the nucleus. If this is
true, WHY1 may have a function in plant adaptation to oxidative
stress (Lepage et al., 2013). Second, WHY1 might be
sequestered at a certain time point or at a modified situation into
the nucleus as a transcription factor. The first evidence was
provided from studies using a recombinant form of WHY1, which
was able to translocate from transplastomic chloroplasts to the
nucleus (Isemer et al., 2012). Repression of downstream gene
WRKY53 by WHY1 in the nucleus was dependent on the
developmental stage (Miao et al., 2013). Analysis of the WHY1
protein sequence revealed 18 potential phosphorylation sites (13
serines and 5 threonines) at regions between positions 19 and 24,
positions 107 and 152, and positions 296 and 303. In this study,
we found that WHY1 phosphorylation status in the nucleus was
increased with plant aging (Figure 1C). By using a biochemical
and genetic approach, we identified a WHY1 interacting partner,
CIPK14, which was located in both the cytoplasm and nucleus
(Figure 2). CIPK14 was able to phosphorylate WHY1 and
Phosphorylation of WHY1 by CIPK14 Affects Its Dual Functions
enhanced nucleus accumulation of phosphorylated WHY1
(Figure 3). The increased phosphorylation of the nuclear isoform
WHY1 protein promoted the binding of WHY1 to the promoter of
WRKY53, which led to decreased expression of WRKY53 at the
early leaf senescence stage (at week 5–6) and SAG12 gene
expression, resulting in the stay-green phenotype as expected.
However, in plants overexpressing CIPK14, only 95% of stay-
green phenotype appeared and the remaining 5% showed a
variegation pale-green phenotype (Figure 4). This 5% pale-green
phenotype had decreased whole-plastid ribosomal RNA, hinting
at ribosome deficiency, while the 95% stay-green plants only
exhibited a partial defect in the biogenesis of the large ribosomal
subunit (16S, 23S, RPL, and RPS); albeit both of them showed
decreased plastid isoform of WHY1 proteins (Figure 5). These
observations were similar to the ore4-1 mutant in which reduced
RPS17 mRNA and 16S rRNA level and extended leaf longevity
were described by Woo et al. (2002). The T-DNA insertion line of
CIPK14 showed a lethal phenotype (serious early senescence
and cell death), whereas knocking down CIPK14 not only
upregulated the expression of senescence-related WRKY53,
SAG12, WRKY33, and NDHF genes but also downregulated the
expression of MER11 and RAD50 genes known to be involved in
plastid DNA double-strand break DNA repair (Vannier et al.,
2006; Ronceret et al., 2009) as well as downregulated the
expression of photosystem-related NDHF and POR genes
(Figure 7). Interestingly, either overexpressing or knocking down
CIPK14 promoted the expression of cell-death-related genes
PDF1.2a, PDF1.2b, PDF1-3, and SAG101, likely an indirect
effect. Thus, we have identified that CIPK14 affected WHY1 dual
localization and dual function in plastids and in the nucleus and
determined the ratio of nWHY1/pWHY1 for its functioning in
leaves of Arabidopsis.
Molecular Plant
Figure 7. WHY1 Downstream Gene Expres-
sion by Quantitative RT–PCR in CIPK14
Mutants.
qRT–PCR analyses of downstream related
genes of WHY1 in CIPK14 mutants, including
the DSB-related genes and plastid rRNA
genes (A), the senescence-related and cell
death-related genes (B). The relative gene
expression level was normalized to Actin, with
the wild-type as 1. The standard error bars
present three biological replicates and three
technical replicates, n = 3 3 3 = 9; the values
are shown as means ± SD. Asterisks (*P < 0.05,
**P < 0.01, ***P < 0.001) show significant differ-
ences compared with Col-0 wild-type according to
Student’s t test. oe5:oeCIPK14-5; oe6:oeCIPK14-6;
kd1:kdCIPK14-1; kd4:kdCIPK14-4.
Protein phosphorylation is a post-
transcriptional modification critical for pre-
cise functions. Intriguingly, plant SNF1-
related protein kinase 1 (SnRK1) modulates
transcriptional, metabolic, and develop-
mental processes (Cho et al., 2012, 2016).
The SnRK kinase family could physically
and functionally interact with a group of
Ca2+ sensors, Calcineurin B-like (CBL)
proteins (Luan, 2009). Increasing evidence
has highlighted the dynamic localization of CIPKs as
determined by their specific CBL partners, in alternative CBL–
CIPK complexes at either the plasma membrane or the
tonoplast (Waadt et al., 2008; Tang et al., 2015) or other
subcellular compartments (Batistic et al., 2010). The CBL–CIPK
regulatory pathways involved in plants responded to
environmental stresses, to general and ionic stresses in
particular (Luan, 2009; Tang et al., 2015). CIPK14 is one of the
serine-threonine SNF1-related protein kinases, which was previ-
ously reported to interact with a calcium-binding protein,
AtCBL2. Accumulation of AtCBL2 transcripts is activated differ-
entially upon treatment with light and cytokinins (Chikano et al.,
2001; Nozawa et al., 2001, 2003; Hrabak et al., 2003; Akaboshi
et al., 2008). CIPK14 appears to have roles in regulating the
responses of Arabidopsis to ABA and salt stress (Qin et al.,
2008) and is involved in PhyA-mediated FR inhibition of seedling
greening (Qin et al., 2010). Knockout of CIPK14 displayed
etiolated seedlings under far-red light after transfer to white light
(Qin et al., 2010). In our experiment, similarly, loss-of-function in
CIPK14 showed yellow seedlings, even resulting in a lethal
phenotype. Knocking down CIPK14 facilitated the accumulation
of plastid WHY1 proteins coupled with dephosphorylated nuclear
WHY1. Phenotypically, plants of CIPK14 knockdown had fewer
rosette leaves, early bolting and flowering, and were early senes-
cent or even lethal. However, the phenotype of the CIPK14
knockdown mutant could not be recovered by complementation
of plastid WHY1 or nuclear WHY1 protein as we described
above (Figure 6). In fact, our previous result showed that
overexpressing plastid isoform WHY1 mutants caused an
etiolated seedling (Supplemental Figure 4). The decreased
expression of NADPH:protochlorophyllide oxidoreductase
(POR) gene (Figure 7), which was encoded by a key enzyme in
Molecular Plant 10, 749–763, May 2017 ª The Author 2017. 757
Molecular Plant Phosphorylation of WHY1 by CIPK14 Affects Its Dual Functions
Figure 8. CIPK14 Level Control WHY1 Subcellular Localization
and Its Prominent Functions in Plastids and the Nucleus,
Providing a Model for Retrograde Signaling between Plastids
and the Nucleus at the Early Leaf Developmental Stage in
Arabidopsis.
WHIRLY1 has different functions in plastid and the nucleus. The ratio of
nWHY1/pWHY1 is determined by the CIPK14 expression level in the cell.
The phosphorylation status of WHY1 by CIPK14 kinase affects the binding
affinity of WHY1 protein with the promoter of the downstream gene to
control gene expression and the phenotype. Therefore, CIPK14 plays a
key role in switching the roles of WHY1 in the nucleus and plastids.
Impairment of CIPK14 leads to seedling de-greening and plastid damage
due to the distribution of WHY1 in plastids and the nucleus. The impair-
ment of CIPK14 leads to a preference for senescence initiation due to the
phosphorylation status of WHY1 in the nucleus.
chlorophyll biosynthesis in higher plants by knocking down
CIPK14 might explain the phenotype as an indirect senescence
and cell death effect. This was likely to be associated with PhyA
and/or CIPK14-mediated FR inhibition of greening chlorophyll
accumulation, since POR catalyzed the light-dependent reduction
of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) and
played a pivotal role in chlorophyll biosynthesis in higher plants.
Severe repression of POR genes was coupled with irreversible
plastid damage (Qin et al., 2010). In contrast, transcripts
encoding photosynthetic proteins, including NDHF (Figure 7), the
thylakoid NADH dehydrogenase, cytochrome b/f complexes
(Zapata et al., 2007), and chloroplast ribosomes were more
abundant in the WHY1-deficient leaves than those in the wild-
type (Comadira et al., 2015). On the other hand, a few reports
have revealed that protein phosphorylation of transcription
factors affected their DNA-binding affinity at the promoter of
downstream target genes and thus their target gene
expressions. For example, MEKK1-phosphorylated WRKY53
increased the binding affinity of WRKY53 at its own promoter
and activated WRKY53 gene expression, resulting in an early
leaf senescence phenotype (Miao et al., 2007); MPK3/MPK6-
758 Molecular Plant 10, 749–763, May 2017 ª The Author 2017.
phosphorylated WRKY33 enhanced the binding affinity on ACS
gene promoters or its own promoter and mediated the ethylene
response or cell death (Zhou et al., 2009; Mao et al., 2011; Li
et al., 2012). Likewise, AHK3 was responsible for the
phosphorylation of ARR2, which was involved in cytokinin-
mediated control of leaf longevity (Kim et al., 2006). Our data
suggested that knocking down CIPK14 not only decreased
nWHY1 accumulation (Figure 5) but also decreased the
phosphorylation of nWHY1 (Figure 3). The dephosphorylated
nWHY1 had a lower binding affinity with the promoter of the
WRKY53 gene (Figure 3), which promoted WRKY53 transcription
and then enhanced SAG12 gene expression, resulting in an early
senescence phenotype (Figure 4). However, complementation of
nWHY1 in the kdCIPK14 line did not recover the senescent
phenotype and decreased the expression of the WRKY53 gene,
but the expression of SAG12 was more pronounced (Figure 6).
This indicated that only the phosphorylated nWHY1 is the
prominent limitation factor for WRKY53-mediated leaf
senescence initiation at the early senescence stage (at week
5–6), and likely WRKY33-mediated abiotic stress or biotic stress-
related senescence and cell death at the late stage (at week 6–8).
Although phosphorylated nWHY1 was increased with the
progress of plant senescence but WRKY53 expression was
downregulated after senescence initiation at the late senescence
stage (Hinderhofer and Zentgraf, 2001), this was not fully
consistent with the expression pattern of WRKY53 during leaf
development. This could be explained by the fact that the
phosphorylated nWHY1 also enriched at the WRKY33 promoter
(Figure 3D and Miao et al., 2013) or at other WHY1 target genes,
and was even involved in regulation of chromatin remodeling
(Janack et al., 2016). Therefore, CIPK14 might be an upstream
regulator of multiple cell senescence and cell death phenomena.
On the other hand, as for WRKY53, many other factors such as
MEKK1 (Miao et al., 2007), AD protein (Miao et al., 2008), UPL5
(Miao and Zentgraf, 2010), ESR (Miao and Zentgraf, 2007), REV
(Xie et al., 2014), and HDA9 (Chen et al., 2016) all co-regulated
WRKY53 expression. Further molecular analyses of the effects of
CIPK14-mediated nWHY1 phosphorylation on the expression of
target genes of WHY1 may provide new insight into the fine-tune
mechanism of cell senescence initiation in future.
Taken together, this study demonstrates that WHIRLY1 has
different functions in plastids and the nucleus, and the ratio of
nWHY1/pWHY1 is determined by the CIPK14 expression level
in the cell. Phosphorylation of WHY1 by CIPK14 kinase affects
the binding affinity of WHY1 protein to the promoter of down-
stream genes, thus controlling gene expression and giving rise
to the relevant phenotype. Therefore, CIPK14 plays a key role
in switching the roles of WHY1 in the nucleus and plastids, anal-
ogous to the function of SNF1-KIN10 (Baena-Gonzale et al.,
2007; Mathur et al., 2011; Blanco et al., 2014; Ng et al., 2013a).
Moreover, light conditions (Qin et al., 2010), sugar (Lee et al.,
2005; Yan et al., 2014), or a cytokinin (Gan and Amasino, 1995)
or calcium-calmodulin signal (Akaboshi et al., 2008), which
promote CIPK14 expression, might control the subcellular
localization and prominent functions of dual-located WHY1
proteins. Indeed, the CIPK14 expression level and CIPK14
protein level also increased during plant aging (Supplemental
Figure 5), further revealing that their functions might provide
insights into retrograde signaling between plastid and nucleus
(Figure 8).
Phosphorylation of WHY1 by CIPK14 Affects Its Dual Functions
It has become increasingly clear that retrograde signaling probably
includes, at least, factors associated with MAP kinase or hormone,
the WHIRLY family, and SNF1 kinase. Most of these factors appear
to integrate the signals emitted by mitochondria (Law et al., 2015)
or chloroplasts and might represent just the founder members of
much larger signaling networks associated with retrograde
signaling. Moreover, in most cases plastid signals seem to
involve more than a simple signal that moves directly from the
inside of the organelle to the nucleus (Woo et al., 2016). Thus,
organelle dysfunction could induce specific transcription factors
re-localized to the ER (DeClercq et al., 2013; Ng et al., 2013a,
2013b), or from the plastid (WHY1) or mitochondrion (WHY2) into
the cytoplasm or the nucleus, and consequently, enabling them
either to serve as signaling intermediaries between organelles
and the nucleus or to act directly on nuclear genes.
METHODS
Plant Materials
Plants of Arabidopsis thaliana, ecotype Columbia, and a series of
CIPK14 mutant plants were grown in a growth chamber at 22 C with
16 h of illumination under low light conditions (70 mmol s 1 m 2). Under
these conditions, wild-type plants bolted at week 5–6 and developed
flowers within 11–12 weeks; mature seeds could be harvested after
12–13 weeks. Colored threads were used to label entire rosette leaves after
their emergence as previously described (Hinderhofer and Zentgraf, 2001).
Chlorophylls were extracted from whole rosettes with hot methanol in the
presence of magnesium hydroxide carbonate. Chlorophyll concentrations
were determined according to Lichtenthaler (1987).
T-DNA insertion lines SALK_009699 (CIPK14-1), SALK_147899, and
SALK_149471 (CIPK14-2) for CIPK14 were provided by NASC. They
were confirmed by PCR with the primers suggested by the T-DNA Express
tool of the SALK institute and by RT–PCR with gene-specific primers
(Supplemental Figure 2). SALK_147899 and SALK_149471 lines could
no longer generate seeds.
Overexpression of CIPK14, CIPK14 RNAi, and a Series of
Double Mutants of CIPK14 and WHY1
The full-length coding sequence of CIPK14 and a 198 bp fragment
sequence were amplified by PCR using cDNA U09142, cloned into
pENTR/TOPO Gateway vector and sequenced to verify PCR product
sequences. After transfer of the full-length CIPK14 and 198 bp fragment
constructs to the binary destination vectors pB7YWG2.0 and
pB7GWIWGW2(I) (Karimi et al., 2002), respectively, PWHY1-HA
plants (Miao et al., 2013) and wild-type were transformed using
agrobacterium-mediated flower transformation with the vacuum
infiltration procedure (Miao et al., 2004). The seedlings grown from the
collected seeds of the infiltrated plants were selected by hygromycin.
The oepWHY1-HA plasmid with the WHY1-HA protein only located in
plastid and the oenWHY1-HA plasmid with the WHY1 protein only located
in the nucleus were provided by the Krupinska group (Isemer et al., 2012).
The ID-nWHY1-HA plasmid with the WHY1 protein only located in the nu-
cleus under estradiol-inducible promoter was prepared according to Miao
et al. (2013). Three plasmids were transformed into oeCIPK14 and
kdCIPK14 lines as well as wild-type plants according to the above
procedure. The seedlings grown from the collected seeds of the
infiltrated plants were selected by spraying with 0.1% (w/v) glufosinate
ammonium (Basta, Bayer Crop Science, Germany). Western blotting
with HA antibody detected positive double transgenic plants
(Supplemental Figure 3), and screened the second generation (T2) with
a 3:1 ratio to get homozygous transgenic plants.
Molecular Plant
mRNA Preparation, Quantitative RT–PCR, and Northern Blot
Analyses
mRNA was extracted from entire rosettes of Arabidopsis using a
Chemagic mRNA Direct Kit according to the manufacturer’s protocol
(Chemagen, Germany). First-strand cDNA was synthesized using the
qScript cDNA SuperMix Kit according to the manufacturer’s protocol
(Quanta Biosciences, VWR, USA). Gene-specific primers were developed
for the genes SAG12, WRKY33, WRKY53, WHY1, 2, 3, and WHY1
downstream-targeted genes as well as for ACTIN2 as an internal control.
Primer pairs are referred to Miao et al. (2013); other primer sequences are
shown in Supplemental Table 1. The expression of the genes was
monitored by applying 1 mL of the diluted cDNA in an SYBR Green Real-
Time PCR analysis using the qPCR SYBR Green Kit (Quanta Biosciences,
VWR, USA) according to the manufacturer’s protocol. Data analysis
was accomplished using iCycler (Bio-Rad). To calculate PCR efficiencies,
four different cDNA dilutions were applied. To determine the relative
expression rate, data were normalized to the expression level in wild-
type, and these basic points were set to 1; the relative expression formula
described by Pfaffl (2001) was used. Each value represents three technical
replicates of three biological replicates (nine measurements altogether).
Northern blotting was done as described by Melonek et al. (2010) and
Zhang et al. (2016); 1 mg, 5 mg, and 15mg of RNAs were used to do dots
blot, 3 mg of RNA was used to do northern blots; the probes 16S, 23S,
4.5S, 5S rRNA, RPL, and RPS were labeled by 32P via a T4 ligase
assay. Hybridization probes were prepared from the PCR fragments of
the following genes of the chloroplast genome (GenBank: AP000423):
16S (101406–101866), 23S (104691–105063), 23S (107296–107480),
4.5S (107587–107729), and 5S (107926–108087) rRNAs.
Production of Recombinant CIPK14 and CIPK14m168T/D
Protein
The CIPK14 sequence was cloned into the Gateway protein expression
vector pDESTTM17 containing a 6xHis tag for purification (Invitrogen).
CIPK14m168T/D clones were produced with a site-mutagenesis kit
(Strategies Company) according to the manufacturer’s protocol. The
CIPK14cds 168 position amino acid was replaced with Thr of Asp by
designing the primer. The recombinant protein was expressed in the Es-
cherichia coli strain BL21-S1 and purified by nickel NTA affinity chroma-
tography and dialyzed according to the manufacturer’s protocol. The
recombinant WHY1 protein was isolated using the method described by
Miao et al. (2013).
In Vitro DNA-Binding Assays
EMSAs were performed essentially as described by Miao et al. (2004, 2007),
but 32P was labeled by T4 ligase. To test the influence of phosphorylation of
WHY1 on its DNA-binding activity, the phosphorylation reaction of WHY1
proteins was performed by incubation of 1 mg of purified recombinant
CIPK14 protein or the CIPK14m168T/D protein and 400 ng of purified
recombinant WHY1 protein with kinase buffer (25 mM Tris [pH 7.5],
10 mM MgCl2 containing 0.135 mM NaVO4, 2 mM DTT, 50 mM ATP, and
0.05 mg/mL aprotinin, 0.5 mM leupeptin) and 0.02 pmol of a [g-32P]ATP-
labeled single-stranded DNA fragment in a total volume of 20 mL at 30 C
for 0, 15, 30, and 45 min. Subsequently, 5 mL of 100 mM EDTA was added
to stop the reaction. The phosphorylated proteins were analyzed on 5%
(w/v) polyacrylamide gels under non-denaturing conditions to perform
the DNA-binding assay. To show the specificity of the DNA–protein interac-
tion, a 20-, 50-, or 100-fold excess of the unlabeled DNA fragment was
added. Alternatively, 3 mg of crude nuclear extracts from oeCIPK14/
PWHY1-HA, kdCIPK14/PWHY1-HA, and wt/PWHY1-HA plants was used
instead of recombinant protein CIPK14 and WHY1 protein to perform the
same assay.
Yeast Two-Hybrid System
The activation domain (33 amino acids) of full-length WHY1 cDNA was
deleted (DWHY1), and the truncated cDNA was cloned into the bait
Molecular Plant 10, 749–763, May 2017 ª The Author 2017. 759
Molecular Plant Phosphorylation of WHY1 by CIPK14 Affects Its Dual Functions
pGBKT7 vector, which harbors the Trp1 selection marker, and was trans- containing 50 mCi [g-32P]ATP (5000 Ci/mmol) and one of the substrates
formed to yeast strain Y187. The cDNA expression library was prepared at a final protein concentration of 0.1 mg/mL. Casein (43 kDa) was used
from 5- and 7-week-old rosette leaves of Arabidopsis and cloned into as a control substrate. CKII was used as a positive kinase control. The
pGADT7-Rec vector containing the GAL4 activation domain and the reaction was terminated by the addition of 53 SDS sample loading buffer.
leu2 selection marker. The yeast strain AH109 was used to express this After electrophoresis on 8% (w/v) SDS–PAGE, the phosphorylated sub-
cDNA library. The two-hybrid screenings and assay were performed via strates were visualized by autoradiography (Miao et al., 2007).
the matting protocol as described in CLONTECH’s Matchmaker GAL4
two-hybrid system 3 and libraries user manual (Clontech/Biosciences, ChIP–qPCR Assay
Palo Alto, CA, USA). For confirmation of the protein–protein interaction,
ChIP assays were performed using the method described by Miao et al.
the full-length CIPK14 and DWHY1 cDNAs were cloned into the prey
(2013). A total of 1.5 g of leaf tissue from entire rosettes of 5-week-old
pGADT7 vector, which harbors the leu2 selection marker, and the bait
plants harboring oeCIPK14, kdCIPK14 in an estradiol-induced promoter
pGBKT7 vector, which contains the Trp1 selection marker, respectively.
fused to nWHY1-HA (ID-nWHY1-HA) background transgenic lines and
If WHY1 was used as bait construct in pGBKT7, the truncated version of
ID-nWHY1 line were treated with 20 mM estradiol after 4 h. The nWHY1
WHY1 (without activated domain) was inserted. The two-hybrid assays
protein was isolated, and induction was confirmed by western blotting.
were performed as described in CLONTECH’s MATCHMAKER GAL4
The fold enrichment was calculated by the ChIP/input ratio and then
Two- Hybrid System 3 Protocol to confirm the interaction.
normalized to the ID-nWHY1 line. Two biological replicates and three
technical replicates were carried out (n = 2 3 3).
Bimolecular Fluorescence Complementation Assay
The full-length WHY1 cDNA with a c-myc tag was cloned into pGFPn173c
vector (Miao et al., 2007) encoding the N-terminal part of the GFP protein
SUPPLEMENTAL INFORMATION
Supplemental Information is available at Molecular Plant Online.
and was sequenced. The full-length CIPK14 cDNA fragment with an HA
tag was cloned into the pGFPc155c vector encoding the C-terminal part
of the GFP protein under the CaMV35S promoter. All appropriate FUNDING
plasmids were isolated using a Maxi kit and transformed to Arabidopsis This work was supported by a grant from the German Research Founda-
protoplast by PEG assay (ZMBP, Transformation Center, Tuebingen, tion (MI1392/1-1) and a grant from the Natural Science Foundation of
Germany) and onion epidemic cell by biolist (Krause et al., 2005). China (31470383).
Protein expression was examined 2–48 h after transformation by
western blot analyses. GFP-dependent fluorescence was observed after
AUTHOR CONTRIBUTIONS
transformation for 2, 4, 8, 16, and 24 h incubation by using a confocal
Y.M. designed the research; Y.M. performed yeast two hybridization,
laser-scanning microscope (Leica SP8). The laser settings were as fol-
northern dot blotting, EMSA, and CoIP; Y.R. constructed the oeCIPK14
lows: 488 nm at 37% of maximal power and 543 nm at 100% of maximal
and kdCIPK14 plasmids, screened mutants, and performed qRT–PCR;
power. Photomultiplicator (PMT) 1 was set by using a 500- to 530-nm win-
Y.L. performed the bimolecular fluorescence complementation assay,
dow to collect the GFP fluorescence. For each construct, at least three
protein localization, and western blotting; Y.J. screened single and double
different independently transformed Arabidopsis protoplast batches
mutants and performed the phenotype analyses and ChIP–qPCR; B.W.
and four different independently transformed onion epidemic cells were
performed in-gel kinase and CIPK14 protein site mutagenesis. B.W. and
analyzed.
Y.M. analyzed the data and wrote the paper.

Co-immunoprecipitation of Plastid and Nuclear Proteins

The plastid and nuclear protein extracted from oeCIPK14-GFP/PWHY1-HA
lines were incubated with the HA antibody at 4 C for 1 h on a rotating
wheel. After addition of protein G beads, the mixture was incubated likewise
for 1 h and then centrifuged at 7000 rpm for 20 s. Protein G beads were
washed three times with buffer (50 mM Tris–HCl, including 0.1% Nonidet
P-40 [pH 8.0]) and then resuspended in SDS–PAGE loading buffer.
Isolation and Immunoblot Analysis of Plastid and Nuclear
Proteins
Chloroplasts were prepared and purified on Percoll step gradients as orig-
inally described by Gruissem et al. (1986) and modified by Grabowski et al.
(2008). Nuclei were isolated as described by Kanazawa et al. (2000).
Proteins were separated on 14% (w/v) acrylamide gels, transferred to
nitrocellulose membranes, and immunodetected using standard proto-
cols. A monoclonal antibody directed toward the HA tag (anti-HA) or the
6xHis tag was purchased from Roche Diagnostics (Mannhein, Germany);
GFP was from Sigma. Pi-Thr168 antibody was provided by the Biochem-
istry Department of Tuebingen University. Antibodies against cytochrome
b559 apoprotein A (Vallon et al., 1987) and histone H2B (Cell Signaling,
Munich, Germany) were used to monitor the purity of the nuclear and
chloroplast fractions, respectively. Western blot analyses were done
according to Miao et al. (2013).
In Vitro Phosphorylation Assay
Phosphorylation activity of CIPK14 and CIPK14m168T/D was assayed at
room temperature for 30 min in a final volume of 20 mL of reaction buffer
(20 mM Tris–HCl [pH 7.5], 1 mM DTT, 10 mM MgCl2, and 100 mM ATP)
ACKNOWLEDGMENTS
We acknowledge Prof. Karin Krupinska of University of Kiel for providing
oepWHY1-HA and oenWHY1-HA plasmid. The antibody against Pi-
Thr168 peptide was provided by the Biochemistry Department of Tuebin-
gen University. No conflict of interest declared.
Received: November 3, 2016
Revised: March 23, 2017
Accepted: March 24, 2017
Published: April 11, 2017
REFERENCES
Akaboshi, M., Hashimoto, H., Ishida, H., Saijo, S., Koizumi, N., Sato,
M., and Shimizu, T. (2008). The crystal structure of plant-specific
calcium-binding protein AtCBL2 in complex with the regulatory
domain of AtCIPK14. J. Mol. Biol. 377:246–257.
Avendano-Vazquez, A.O., Cordoba, E., Llamas, E., San Roman, C.,
Nisar, N., De la Torre, S., Ramos-Vega, M., Gutierrez-Nava, M.D.,
Cazzonelli, C.I., Pogson, B.J., et al. (2014). An uncharacterized
apocarotenoid-derived signal generated in zeta-carotene desaturase
mutants regulates leaf development and the expression of
chloroplast and nuclear genes in Arabidopsis. Plant Cell 26:2524–2537.
Baena-Gonzalez, E., Rolland, F., Thevelein, J.M., and Sheen, J. (2007).
A central integrator of transcription networks in plant stress and energy
signaling. Nature 448:938–942.
Batistic, O., Waadt, R., Steinhorst, L., Held, K., and Kudla, J. (2010).
CBL-mediated targeting of CIPKs facilitates the decoding of calcium
signals emanating from distinct cellular stores. Plant J. 61:211–222.

760 Molecular Plant 10, 749–763, May 2017 ª The Author 2017.
Phosphorylation of WHY1 by CIPK14 Affects Its Dual Functions
Blanco, N.E., Guinea-Diaz, M., Whelan, J., and Strand, A. (2014).
Interaction between plastid and mitochondrial retrograde signalling
pathways during changes to plastid redox status. Philos. Trans. R.
Soc. Lond. Ser. B. Biol. Sci. 369:20130231.
Cappadocia, L., Maréchal, A., Parent, J.S., Lepage, E., Sygusch, J.,
and Brisson, N. (2010). Crystal structures of DNA-Whirly complexes
and their role in Arabidopsis organelle genome repair. Plant Cell
22:1849–1867.
Cappadocia, L., Parent, J.S., Zampini, E., Lepage, E., Sygusch, J., and
Brisson, N. (2012). A conserved lysine residue of plant Whirly proteins
is necessary for higher order protein assembly and protection against
DNA damage. Nucleic Acids Res. 40:258–269.
Chen, X.S., Lu, L., Mayer, K., Scalf, M., Qian, S., Lomax, A., Smith, L.,
and Zhong, X. (2016). POWERDRESS interacts with HISTONG
DEACETYLASE 9 to promote aging in Arabidopsis. Elife 5:e17214.
Chikano, H., Ogawa, M., Ikeda, Y., Koizumi, N., Kusano, T., and Sano,
H. (2001). Two novel genes encoding SNF-1 related protein kinases
from Arabidopsis thaliana: differential accumulation of AtSR1 and
AtSR2 transcripts in response to cytokinins and sugars, and
phosphorylation of sucrose synthase by AtSR2. Mol. Gen. Genet.
264:674–681.
Cho, Y.H., Hong, J.W., Kim, E.C., and Yoo, S.D. (2012). Regulatory
functions of SnRK1 in stress-responsive gene expression and in
plant growth and development. Plant Physiol. 158:1955–1964.
Cho, Y.H., Wen, T.N., Wang, Y.T., and Shih, M.C. (2016). Quantitative
phosphoproteomics of protein kinase SnRK1 regulated protein
phosphorylation in Arabidopsis under submergence. J. Exp. Bot.
67:2745–2760.
Comadira, G., Rasool, B., Kaprinska, B., Garcı́a, B.M., Morris, J.,
Verrall, S.R., Bayer, M., Hedley, P.E., Hancock, R.D., and Foyer,
C.H. (2015). WHIRLY1 functions in the control of responses to
nitrogen deficiency but not aphid infestation in barley. Plant Physiol.
168:1140–1151.
DeClercq, I., Vermeirssen, V., Van Aken, O., Vandepoele, K., Murcha,
M.W., Law, S.R., Inze, A., Ng, S., Ivanova, A., Rombaut, D., et al.
(2013). The membrane-bound NAC transcription factor ANAC013
functions in mitochondrial retrograde regulation of the oxidative
stress response in Arabidopsis. Plant Cell 25:3472–3490.
Desveaux, D., Després, C., Joyeux, A., Subramaniam, R., and Brisson,
N. (2000). PBF-2 is a novel single-stranded DNA binding factor
implicated in PR-10a gene activation in potato. Plant Cell 12:1477–
1489.
Desveaux, D., Allard, J., Brisson, N., and Sygusch, J. (2002). A new
family of plant transcription factors displays a novel ssDNA-binding
surface. Nat. Struct. Biol. 9:512–517.
Desveaux, D., Subramaniam, R., Després, C., Mess, J.N., Lévesque,
C., Fobert, P.R., Dangl, J.L., and Brisson, N. (2004). A ‘‘Whirly’’
transcription factor is required for salicylic acid-dependent disease
resistance in Arabidopsis. Dev. Cell 6:229–240.
Foyer, C.H., Karpinska, B., and Krupinska, K. (2014). The functions of
WHIRLY1 and REDOX-RESPONSIVE TRANSCRIPTION FACTOR 1 in
cross tolerance responses in plants: a hypothesis. Philos. Trans. R.
Soc. Lond. B. Biol. Sci. 369:20130226.
Gan, S., and Amasino, R.M. (1995). Inhibition of leaf senescence by
autoregulated production of cytokinin. Science 270:1986–1988.
Grabowski, E., Miao, Y., Mulisch, M., and Krupinska, K. (2008). Single-
stranded DNA-binding protein Whirly1 in barley leaves is located in
plastids and the nucleus of the same cell. Plant Physiol. 147:1800–
1804.
Gruissem, W., Greenberg, B., Zurawski, G., and Hallick, R. (1986).
Chloroplast gene expression and promoter identification in
chloroplast extracts. Methods Enzymol. 118:253–270.
Molecular Plant
Guo, Y., Halfter, U., Ishitani, M., and Zhu, J.K. (2001). Molecular
characterization of functional domains in the protein kinase SOS2
that is required for plant salt tolerance. Plant Cell 13:1383–1400.
Hinderhofer, K., and Zentgraf, U. (2001). Identification of a transcription
factor specifically expressed at the onset of leaf senescence. Planta
213:469–473.
Hrabak, E.M., Chan, C.W., Gribskov, M., Harper, J.F., Choi, J.H.,
Halford, N., Kudla, J., Luan, S., Nimmo, H.G., Sussman, M.R.,
et al. (2003). The Arabidopsis CDPK-SnRK superfamily of protein
kinases. Plant Physiol. 132:666–680.
€ fer, A., Kirchner, S., Koop, H.U., and
Isemer, R., Mulisch, M., Scha
Krupinska, K. (2012). Recombinant Whirly1 translocates from
transplastomic chloroplasts to the nucleus. FEBS Lett. 586:85–88.
Janack, B., Sosoi, P., Krupinska, K., and Humbeck, K. (2016).
Knockdown of WHIRLY1 affects drought stress-induced leaf
senescence and histone modifications of the senescence-associated
gene HvS40. Plants (Basel) 5:37.
Kanazawa, S., Sano, H., and Koshiba, T. (2000). Changes in
antioxidative enzymes in cucumber cotyledons during natural
senescence: comparison with those during dark-induced
senescence. Physiol. Plant 109:211–216.
Karimi, M., Inzé, D., and Depicker, A. (2002). GATEWAY vectors for
Agrobacterium-mediated plant transformation. Trends Plant Sci.
7:193–195.
Kim, H.J., Ryu, H., Hong, S.H., Woo, H.R., Lim, P.O., Lee, I.C., Sheen,
J., Nam, H.G., and Hwang, I. (2006). Cytokinin-mediated control
of leaf longevity by AHK3 through phosphorylation of ARR2 in
Arabidopsis. Proc. Natl. Acad. Sci. USA 103:814–819.
Kleine, T., and Leister, D. (2016). Retrograde signaling: organelles go
networking. Biochim. Biophys. Acta 1857:1313–1325.
Kleine, T., Voigt, C., and Leister, D. (2009). Plastid signalling to the
nucleus: messengers still lost in the mists? Trends Genet. 25:185–192.
Koussevitzky, S., Nott, A., Mockler, T.C., Hong, F., Sachetto-Martins,
G., Surpin, M., Lim, J., Mittler, R., and Chory, J. (2007). Signals from
chloroplasts converge to regulate nuclear gene expression. Science
316:715–719.
Krause, K., and Krupinska, K. (2009). Nuclear regulators with a second
home in organelles. Trends Plant Sci. 14:194–199.
€ diger, A., Scha
Krause, K., Kilbienski, I., Mulisch, M., Ro € fer, A., and
Krupinska, K. (2005). DNA-binding proteins of the Whirly family in
Arabidopsis thaliana are targeted to the organelles. FEBS Lett.
579:3707–3712.
€ hnhardt, D., Fischer-Kilbienski, I., Kucharewicz, W.,
Krupinska, K., Da
Scharrenberg, C., Trosch, M., and Buck, F. (2014). Identification of
WHIRLY1 as a factor binding to the promoter of the stress- and
senescence-associated gene HvS40. J. Plant Growth Regul.
33:91–105.
Law, Y.S., Zhang, R., Guan, X., Cheng, S., and Sun, F. (2015).
Phosphorylation and dephosphorylation of the presequence of
pMORF3 during import into mitochondria from Arabidopsis thaliana.
Plant Physiol. 169:1344–1355.
Lee, E.J., Iai, H., Sano, H., and Koizumi, N. (2005). Sugar responsible
and tissue specific expression of a gene encoding AtCIPK14,
an Arabidopsis CBL-interacting protein kinase. Biosci. Biotechnol.
Biochem. 69:242–245.
Lepage, É., Zampini, É., and Brisson, N. (2013). Plastid genome
instability leads to reactive oxygen species production and plastid-
to-nucleus retrograde signaling in Arabidopsis. Plant Physiol.
163:867–881.
Lichtenthaler, H.K. (1987). Chlorophylls and carotenoids: pigments of
photosynthetic biomembranes. Methods Enzymol. 148:350–383.
Molecular Plant 10, 749–763, May 2017 ª The Author 2017. 761
Molecular Plant Phosphorylation of WHY1 by CIPK14 Affects Its Dual Functions
Li, G., Meng, X., Wang, R., Mao, G., Han, L., Liu, Y., and Zhang, S.
(2012). Dual-level regulation of ACC synthase activity by MPK3/
MPK6 cascade and its downstream WRKY transcription factor
during ethylene induction in Arabidopsis. PLoS Genet. 8:e1002767.
Luan, S. (2009). The CBL-CIPK network in plant calcium signaling. Trends
Plant Sci. 14:37–42.
Mao, G., Meng, X., Liu, Y., Zheng, Z., Chen, Z., and Zhang, S. (2011).
Phosphorylation of a WRKY transcription factor by two pathogen-
responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis.
Plant Cell 23 (4):1639–1653.
Maréchal, A., Parent, J.S., Véronneau-Lafortune, F., Joyeux, A.,
Lang, B.F., and Brisson, N. (2009). Whirly Proteins maintain
plastid genome stability in Arabidopsis. Proc. Natl. Acad. Sci. USA
106:14693–14698.
Mathur, S., Vyas, S., Kapoor, S., and Tyagi, A.K. (2011). The Mediator
complex in plants: structure, phylogeny, and expression profiling of
representative genes in a dicot (Arabidopsis) and a monocot (rice)
during reproduction and abiotic stress. Plant Physiol. 157:1609–
1627.
Melonek, J., Mulisch, M., Schmitz-Linneweber, C., Grabowski, E.,
Hensel, G., and Krupinska, K. (2010). Whirly1 in chloroplasts
associates with intron containing RNAs and rarely co-localizes with
nucleoids. Planta 232:471–481.
Miao, Y., and Zentgraf, U. (2007). The antagonist function of Arabidopsis
WRKY53 and ESR/ESP in leaf senescence is modulated by the
jasmonic and salicylic acid equilibrium. Plant Cell 19:819–830.
Miao, Y., and Zentgraf, U. (2010). A HECT E3 ubiquitin ligase negatively
regulates Arabidopsis leaf senescence through degradation of the
transcription factor WRKY53. Plant J. 63:179–188.
Miao, Y., Laun, T.M., Smykowski, A., and Zentgraf, U. (2007).
Arabidopsis MEKK1 can take a short cut: it can directly interact with
senescence-related WRKY53 transcription factor on the protein level
and can bind to its promoter. Plant Mol. Biol. 65:63–76.
Miao, Y., Jiang, J., Ren, Y., and Zhao, Z. (2013). The single-stranded
DNA-binding protein WHIRLY1 represses WRKY53 expression and
delays leaf senescence in a developmental stage-dependent manner
in Arabidopsis. Plant Physiol. 163:746–756.
Miao, Y., Laun, T., Zimmermann, P., and Zentgraf, U. (2004). Targets of
the WRKY53 transcription factor and its role during leaf senescence in
Arabidopsis. Plant Mol. Biol. 55:853–867.
Miao, Y., Smykowski, A., and Zentgraf, U. (2008). A novel upstream
regulator of the WRKY53 transcription during leaf senescence of
Arabidopsis thaliana. Plant Biol. suppl. 10:110–120.
Ng, S., Giraud, E., Duncan, O., Law, S.R., Wang, Y., Xu, L., Narsai, R.,
Carrie, C., Walker, H., Day, D.A., et al. (2013a). Cyclin-dependent
kinase E1 (CDKE1) provides a cellular switch in plants between
growth and stress responses. J. Biol. Chem. 288:3449–3459.
Ng, S., Ivanova, A., Duncan, O., Law, S.R., Van Aken, O., De Clercq, I.,
Wang, Y., Carrie, C., Xu, L., Kmiec, B., et al. (2013b). A membrane
bound NAC transcription factor, ANAC017, mediates mitochondrial
retrograde signaling in Arabidopsis. Plant Cell 25:3450–3471.
Nozawa, A., Koizumi, N., and Sano, H. (2001). An Arabidopsis SNF1-
related protein kinase, AtSR1, interacts with a calcium-binding
protein, AtCBL2, of which transcripts respond to light. Plant Cell
Physiol. 42:976–981.
Nozawa, A., Sawada, Y., Akiyama, T., Koizumi, N., and Sano, H. (2003).
Variable interactions between sucrose non-fermented 1-related protein
kinases and regulatory proteins in higher plants. Biosci. Biotechnol.
Biochem. 67:2533–2540.
Pfaffl, M.W. (2001). A new mathematical model for relative quantification
in real-time RT-PCR. Nucleic Acids Res 29, No.9.
762 Molecular Plant 10, 749–763, May 2017 ª The Author 2017.
Pfalz, J., Liere, K., Kandlbinder, A., Dietz, K.J., and Oelmueller, R.
(2005). pTAC2, -6, and -12 are components of the transcriptionally
active plastid chromosome that are required for plastid gene
expression. Plant Cell 18:176–197.
Prikryl, J., Watkins, K.P., Friso, G., Wijk, K.J., and Barkan, A. (2008). A
member of the Whirly family is a multifunctional RNA- and DNA-binding
protein that is essential for chloroplast biogenesis. Nucleic Acids Res.
36:5152–5165.
Qin, Y., Li, X., Guo, M., Deng, K., Lin, J., Tang, D., Guo, X., and Liu, X.
(2008). Regulation of salt and ABA responses by CIPK14, a calcium
sensor interacting protein kinase in Arabidopsis. Sci. China Life Sci.
51:391–401.
Qin, Y., Guo, M., Li, X., Xiong, X., He, C., Nie, X., and Liu, X. (2010).
Stress responsive gene CIPK14 is involved in phytochrome
A-mediated far-red light inhibition of greening in Arabidopsis. Sci.
China Life Sci. 53:1307–1314.
Ronceret, A., Doutriaux, M.P., Golubovskaya, I.N., and Pawlowski,
W.P. (2009). PHS1 regulates meiotic recombination and homologous
chromosome pairing by controlling the transport of RAD50 to the
nucleus. Proc. Natl. Acad. Sci. USA 106:20121–20126.
Sun, X., Feng, P., Xu, X., Guo, H., Ma, J., Chi, W., Lin, R., Lu, C., and
Zhang, L. (2011). A chloroplast envelope-bound PHD transcription
factor mediates chloroplast signals to the nucleus. Nat. Commun.
2:477.
Tang, R.J., Zhao, F.G., Garciaa, V.J., Kleista, T.J., Yang, L., Zhang,
H.X., and Luan, S. (2015). Tonoplast CBL–CIPK calcium signaling
network regulates magnesium homeostasis in Arabidopsis. Proc.
Natl. Acad. Sci. USA 112:3134–3139.
Vallon, O., Hoyer-Hansen, G., and Simpson, D.J. (1987). Photosystem II
and cytochrome b559 in the stroma lamellae of barley chloroplasts.
Carlsberg Res. Commun. 52:405–421.
Van Aken, O., Zhang, B., Law, S., Narsai, R., and Whelan, J. (2013).
AtWRKY40 and AtWRKY63 modulate the expression of stress-
responsive nuclear genes encoding mitochondrial and chloroplast
proteins. Plant Physiol. 162:254–271.
Vannier, J.B., Depeiges, A., White, C., and Gallego, M.E. (2006). Two
roles for Rad50 in telomere maintenance. EMBO. J. 25:4577–4585.
Waadt, R., Schmidt, L.K., Lohse, M., Hashimoto, K., Bock, R., and
Kudla, J. (2008). Multicolor bimolecular fluorescence complementation
reveals simultaneous formation of alternative CBL/CIPK complexes in
planta. Plant J. 56:505–516.
Woo, H.R., Goh, C.H., Park, J.H., de la Serve, B.T., Kim, J.H., Park, Y.I.,
and Nam, H.G. (2002). Extended leaf longevity in the ore4-1 mutant of
Arabidopsis with a reduced expression of a plastid ribosomal protein
gene. Plant J. 31:331–340.
Woo, H.R., Koo, H.J., Kim, J., Jeong, H., Yang, J.O., Lee, I.H., Jun, J.H.,
Choi, S.H., Park, S.J., et al. (2016). Programming of plant leaf
senescence with temporal and inter-organellar coordination of
transcriptome in Arabidopsis. Plant Physiol. 171:452–467.
Xie, Y., Huhn, K., Brandt, R., Potschin, M., Bieker, S., Straub, D., Doll,
J., Drechsler, T., Zentgraf, U., and Wenkel, S. (2014). REVOLUTA
and WRKY53 connect early and late leaf development in
Arabidopsis. Development 141:4772–4783.
Xiong, J.Y., Lai, C.X., Qu, Z., Yang, X.Y., Qin, X.H., and Liu, G.Q. (2009).
Recruitment of AtWHY1 and AtWHY3 by a distal element upstream of
the kinesin gene AtKP1 to mediate transcriptional repression. Plant
Mol. Biol. 71:437–449.
Yan, J., Niu, F., Liu, W.Z., Zhang, H., Wang, B., Lan, W., Che, Y., Yang,
B., Luan, S., and Jiang, Y.Q. (2014). Arabidopsis CIPK14 positively
regulates glucose response. Biochem. Biophys. Res. Commun.
450:1679–1683.
Phosphorylation of WHY1 by CIPK14 Affects Its Dual Functions
Yoo, H.H., Kwon, C., Lee, M.M., and Chung, I.K. (2007). Single-stranded
DNA binding factor AtWHY1 modulates telomere length homeostasis
in Arabidopsis. Plant J. 49:442–451.
Zapata, J.M., Guera, A., Esteban-Carrasco, A., Martin, M., and
Sabater, B. (2005). Chloroplasts regulate leaf senescence: delayed
senescence in transgenic ndhF-defective tobacco. Cell Death Differ.
12:1277–1284.
Zapata, J.M., Gasulla, F., Esteban-Carrasco, A., Barreno, E., and
Guera, A. (2007). Inactivation of a plastid evolutionary conserved
gene affects PSII electron transport, life span and fitness of tobacco
plants. New Phytol. 174:357–366.
Molecular Plant
Zhang, Y., Hou, M., and Tan, B. (2013). The requirement of WHIRLY1 for
embryogenesis is dependent on genetic background in maize. PLoS
One 8:e67369.
Zhang, J., Yuan, H., Yang, Y., Fish, T., Lyi, S.M., Thannhauser, T.W.,
Zhang, L., and Li, L. (2016). Plastid ribosomal protein S5 is involved
in photosynthesis, plant development, and could stress tolerance in
Arabidopsis. J. Exp. Bot. 67:2731–2744.
Zhou, C., Cai, Z., Guo, Y., and Gan, S. (2009). An Arabidopsis mitogen-
activated protein kinase cascade, MKK9-MPK6, plays a role in leaf
senescence. Plant Physiol. 150:167–177.
Molecular Plant 10, 749–763, May 2017 ª The Author 2017. 763