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The Os-AKT1 Channel Is Critical for K+ Uptake in Rice Roots
and Is Modulated by the Rice CBL1-CIPK23 Complex
W OPEN
Juan Li,1 Yu Long,1 Guo-Ning Qi,1,2 Juan Li, Zi-Jian Xu, Wei-Hua Wu, and Yi Wang3
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, National Plant Gene Research Centre
(Beijing), China Agricultural University, Beijing 100193, China
ORCID ID: 0000-0002-3660-5859 (Y.W.)
Potassium (K+) is one of the essential nutrient elements for plant growth and development. Plants absorb K+ ions from the
environment via root cell K+ channels and/or transporters. In this study, the Shaker K+ channel Os-AKT1 was characterized
for its function in K+ uptake in rice (Oryza sativa) roots, and its regulation by Os-CBL1 (Calcineurin B-Like protein1) and OsCIPK23 (CBL-Interacting Protein Kinase23) was investigated. As an inward K+ channel, Os-AKT1 could carry out K+ uptake
and rescue the low-K+-sensitive phenotype of Arabidopsis thaliana akt1 mutant plants. Rice Os-akt1 mutant plants showed
decreased K+ uptake and displayed an obvious low-K+-sensitive phenotype. Disruption of Os-AKT1 significantly reduced the
K+ content, which resulted in inhibition of plant growth and development. Similar to the AKT1 regulation in Arabidopsis, OsCBL1 and Os-CIPK23 were identified as the upstream regulators of Os-AKT1 in rice. The Os-CBL1-Os-CIPK23 complex could
enhance Os-AKT1-mediated K+ uptake. A phenotype test confirmed that Os-CIPK23 RNAi lines exhibited similar K+-deficient
symptoms as the Os-akt1 mutant under low K+ conditions. These findings demonstrate that Os-AKT1-mediated K+ uptake in
rice roots is modulated by the Os-CBL1-Os-CIPK23 complex.
INTRODUCTION
Potassium (K+) is the most abundant monovalent cation in plant
cells and plays essential roles in plant growth and development
(Clarkson and Hanson, 1980; Leigh and Wyn Jones, 1984). The
K+ concentration in plant cell cytosol is maintained in the range
of 100 mM (Ashley et al., 2006; Wyn Jones and Pollard, 1983).
This relatively high and stable K+ concentration supports many
physiological processes in plant cells, such as enzyme activation, protein biosynthesis, and membrane potential maintenance
(Marschner, 1995).
Potassium constitutes ;2.5% of the lithosphere and is the fourth
most abundant mineral element in the earth (Sparks and Huang,
1985). However, only the free K+ ions can be absorbed and utilized
by plants. The concentration of free K+ at the surfaces of plant roots
in soils is usually below 1 mM (Luan et al., 2009). Therefore, plants
often suffer the low-K+ stress under natural conditions and display
K+-deficient symptoms, typically leaf chlorosis and inhibition of
growth and development (Mengel and Kirkby, 2001). However,
plants can perceive the K+-deficient condition and adapt to low-K+
stress by altering root morphology, changing the K+ utilization
strategy, and modifying the K+ acquisition mechanism (Schachtman
and Shin, 2007; Wang and Wu, 2013).
1 These
authors contributed equally to this work.
address: Institute of Plant Physiology and Ecology, Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences, 300
Feng Lin Road, Shanghai 200032, China.
3 Address correspondence to [email protected].
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 (www.plantcell.org) is: Yi Wang (yiwang@cau.
edu.cn).
W
Online version contains Web-only data.
OPEN
Articles can be viewed online without a subscription.
www.plantcell.org/cgi/doi/10.1105/tpc.114.123455
2 Current
In plant roots, K+ absorption from soils is mainly mediated by
K+ channels and transporters whose transcription may be induced
and activities may be enhanced in response to K+-deficient
stress (Wang and Wu, 2013). AKT1 (ARABIDOPSIS K +
TRANSPORTER1) has been identified as an inward-rectifying K+
channel in Arabidopsis thaliana and plays crucial roles in K+ uptake
from soil into root cells (Hirsch et al., 1998; Ivashikina et al., 2001;
Lagarde et al., 1996; Spalding et al., 1999). Loss of function of
At-AKT1 leads to a reduction of K+ uptake and makes plants
hypersensitive to low-K+ stress (Hirsch et al., 1998; Spalding
et al., 1999; Xu et al., 2006). AKT1 activity is positively regulated
by CBL1/9-CIPK23 protein complexes in Arabidopsis (Xu et al.,
2006). The calcineurin B-like protein CBL1 and/or CBL9 interacts
with protein kinase CIPK23 at the plasma membrane (PM), where
CIPK23 phosphorylates AKT1 and activates AKT1-mediated K+
uptake (Xu et al., 2006). Several AKT1 orthologs have been
identified in other plant species, such as Os-AKT1 in rice (Oryza
sativa; Fuchs et al., 2005), Zm-ZMK1 in maize (Zea mays; Philippar
et al., 1999), Ta-AKT1 in wheat (Triticum aestivum; Buschmann
et al., 2000), Hv-AKT1 in barley (Hordeum vulgare; Boscari et al.,
2009), Sl-LKT1 in tomato (Solanum lycopersicum; Hartje et al.,
2000), and St-SKT1 in potato (Solanum tuberosum; Zimmermann
et al., 1998). Similar regulatory mechanisms, with CBL-CIPK
complex-modulating K+ channels, were also reported in barley
(Boscari et al., 2009) and grape (Vitis vinifera) plants (Cuéllar et al.,
2010, 2013).
Rice is one of the most important crops and staple foods in
the world, and rice grain yield and quality are significantly dependent on the K+ supply in the soils (Wanasuria et al., 1981;
Dobermann et al., 1996). However, the K+ absorption mechanism
in rice roots is still unclear. Several K+ channels and transporters
have been cloned and considered as candidate components to
mediate K+ uptake in rice roots, including Os-AKT1 (Golldack
et al., 2003; Fuchs et al., 2005), Os-HAK1 (Bañuelos et al., 2002),
The Plant Cell Preview, www.aspb.org ã 2014 American Society of Plant Biologists. All rights reserved.
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Os-HKT2;1 (Horie et al., 2007; Yao et al., 2010), and Os-HKT2;4
(Horie et al., 2011). All of these channel and transporter proteins
show the K+ transport activity in heterogeneous expression systems, but their physiological functions in rice seedlings have not
been characterized. Among these candidates, Os-AKT1 seems to
be the most important component involved in K+ uptake in rice
roots. The previous data showed that Os-AKT1 is mainly expressed
in rice roots (Golldack et al., 2003), and Os-AKT1 transcription
could be repressed by salt stress (Fuchs et al., 2005). Os-AKT1 has
been characterized as an inward-rectifying K+ channel in HEK293
cells and showed high ion selectivity to K+ over Na+ (Fuchs et al.,
2005). In this study, we investigated the physiological function of
Os-AKT1 in rice K+ uptake as well as its regulatory mechanism.
RESULTS
Protein Structure of Os-AKT1
Os-AKT1 shares high similarity with other Shaker K+ channels
from plant species, such as At-AKT1, Sl-LKT1, St-SKT1, ZmZMK1, Ta-AKT1, and Hv-AKT1 (58, 60, 60, 73, 76, and 75%
identities, respectively) (Supplemental Figure 1). Phylogenetic
analysis classified the K+ channels from monocots and dicots
separately (Supplemental Figure 2 and Supplemental Data Set
1). The Os-AKT1 P-loop domain contains a typical TxxTxGYG
motif, a hallmark of K+-selective channels (Doyle et al., 1998),
suggesting that Os-AKT1 is likely to exhibit high ion selectivity
for K+. The high degree of similarity of these Shaker K+ channels
indicates that they likely have similar physiological functions in
the different plant species.
Figure 1. Subcellular Localization and Expression Pattern of Os-AKT1.
Subcellular Localization and Expression Pattern of Os-AKT1
To test the subcellular localization of Os-AKT1, the fusion gene
of Os-AKT1-EGFP was constructed and transformed into tobacco (Nicotiana benthamiana) leaves. CBL1n-OFP was used as
et al., 2008). When Os-AKT1-EGFP was
the PM marker (Batistic
coexpressed with CBL1n-OFP in tobacco leaves, the green and
red fluorescence showed remarkable overlap in tobacco leaves
(Figure 1A), which indicates a PM localization for Os-AKT1 in
plant cells.
To determine the expression profiles of Os-AKT1 in rice,
transgenic rice plants carrying a GUS gene under control of an
Os-AKT1 promoter fragment (1010 bp; O. sativa ssp japonica cv
Nipponbare) were generated. The b-glucuronidase (GUS) activity assays showed that the Os-AKT1 promoter drives strong
expression in roots (Figures 1B and 1C) and slight expression in
shoots (Figure 1B). In root tissues, GUS activity was observed in
all cell types (Figures 1C and 1D). The expression of Os-AKT1
in the epidermis and root hairs suggests a physiological role for
Os-AKT1 in root K+ uptake from soil (Figures 1C and 1D). Furthermore, GUS activity was also detected in cortex, endodermis,
and vascular bundles (Figure 1D), which indicates Os-AKT1 may
also participate in K+ translocation in roots. In shoot tissues,
Os-AKT1 promoter activity was mainly found in epidermis and
vascular bundles (Figures 1E and 1F).
The GUS activity assays showed that the transcription
of Os-AKT1 was not affected after K + deficiency treatment
(A) Transient expression of Os-AKT1-EGFP and CBL1n-OFP in
N. benthamiana leaves. Left panel, GFP image; middle panel, OFP image;
right panel, merge of GFP and OFP image (bar = 50 mm).
(B) to (G) Expression pattern of Os-AKT1 determined in ProOs-AKT1:
GUS transgenic rice. The GUS staining of 10-d-old seedling (B), primary
root (C), transverse section of primary root (D), transverse section of leaf
(E), transverse section of stem (F), and anthers (G). RH, root hair; Ep,
epidermis; Ex, exodermis; Co, Cortex; En, endodermis; Ph, phloem;
X, xylem. Bars = 100 mm.
(Supplemental Figure 3A). Meanwhile, the quantitative real-time
PCR results also confirmed that the low-K+ treatment did not induce Os-AKT1 expression in both roots and shoots (Supplemental
Figures 3B and 3C).
K+ Transport Activity of Os-AKT1 in Yeast
The K+ transport activity of Os-AKT1 was tested in the auxotrophic yeast mutant strain R5421 (trk1D, trk2D) and its wild-type
strain R757 (Gaber et al., 1988; Nakamura et al., 1997). R5421
was defective in K+ uptake and could not grow under low-K+
conditions. Os-AKT1 was transformed into R5421, and AKT1
from Arabidopsis was also introduced into R5421 as a positive
control. The yeast growth assays were performed on AP medium (Rodríguez-Navarro and Ramos, 1984) containing different
concentrations of K+. Under high K+ conditions (50 mM), all
tested yeast strains showed similar growth (Figure 2A). Along
Os-AKT1 Functions in Rice K+ Uptake
with the decline of K+ concentration, the growth of R5421 was
significantly depressed (Figure 2A), and both rice and Arabidopsis AKT1 could rescue the growth defect of R5421 mutant
(Figure 2A), suggesting that the Os-AKT1 channel has a similar
function in K+ uptake as the Arabidopsis AKT1 channel. Both
channels could mediate K+ uptake under low K+ concentrations
(Km AKT1 = 106 mM, Km Os-AKT1 = 114 mM) (Figure 2B).
Functional Characterization of Os-AKT1 in Arabidopsis
AKT1 is an important K+ channel and plays crucial roles in K+
uptake in Arabidopsis roots. The Arabidopsis akt1 mutant plants
exhibit low-K+-sensitive phenotypes and a significant decrease
in K+ content after growth on low-K+ medium (Hirsch et al.,
1998; Spalding et al., 1999; Xu et al., 2006). To examine the
possible function of Os-AKT1, the coding sequence of Os-AKT1
was cloned and transformed into Arabidopsis akt1 mutant (ecotype
Columbia [Col]) and two transgenic lines were obtained. The phenotype tests showed that the low-K+-sensitive phenotype of akt1
mutant was rescued in these two transgenic lines (akt1/Os-AKT1),
which displayed a similar phenotype as wild-type (Col) plants
(Figures 2C and 2D). The K+ contents in these two transgenic
lines were also rescued (Figure 2E). Moreover, the root K+
content in these two lines were even higher than that in wildtype plants under low-K+ conditions (Figure 2E). These results
demonstrate that Os-AKT1 has similar function in K+ uptake as
AKT1 in Arabidopsis.
To measure the Os-AKT1-mediated K+ currents in Arabidopsis root cells, patch-clamp whole-cell recording was conducted using root cell protoplasts. Inward K+ currents were
observed in root cell protoplasts of wild-type plants, but not in
those of the akt1 mutant (Figure 2F), suggesting that the inward
K+ currents in wild-type root cells were contributed by AKT1
channel (Hirsch et al., 1998; Reintanz et al., 2002; Xu et al.,
2006). However, the akt1/Os-AKT1 transgenic line showed obvious inward K+ currents (Figure 2F), suggesting that Os-AKT1
could mediate K+ uptake in Arabidopsis root cells. It should be
noted that the Os-AKT1-mediated K+ currents were different
from the currents conducted by Arabidopsis AKT1 channel
(Figures 2F and 2G). As shown in Figure 2H, the half-activation
voltage of Os-AKT1 (V1/2 = 2173 6 5 mV) was more negative than
that of At-AKT1 (V1/2 = 2133 6 4 mV), suggesting that Os-AKT1
requires a more negative membrane potential to be activated.
Phenotype Analysis of Rice Os-akt1 Mutant
The T-DNA insertion mutant line of Os-AKT1 from RiceGE (Rice
Functional Genomic Express Database, http://signal.salk.edu/cgibin/RiceGE) was used to validate the physiological function of OsAKT1 in rice. In this Os-akt1 mutant, the T-DNA fragment is inserted
into the 59-untranslated region of Os-AKT1, 303 bp upstream of the
start codon (Figure 3A). The insertion leads to the knockdown of
Os-AKT1 in the mutant plants compared with wild-type (O. sativa
ssp japonica cv Dongjin) plants (Figure 3B). The results of DNA gel
blot assays indicated that there is only one copy of the T-DNA
fragment inserting into the mutant plants (Figure 3C).
Using hydroponic culture, the phenotype of Os-akt1 mutant
and wild-type (Dongjin) plants was tested. The 14-d-old rice
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seedlings were transferred into normal (1 mM K+) and low K+
(100 mM K+) medium. After growth for 7 d, the Os-akt1 mutant
seedlings showed overall growth inhibition compared with wildtype plants in both normal and low K+ conditions (Figures 3D,
3F, and 3G). In addition, the Os-akt1 mutant displayed the
brown spots on old leaves, which was a typical K+-deficient
symptom of rice (Figure 3E). This symptom in Os-akt1 mutant
became more remarkable under low K+ conditions (Figure 3E).
The K+ content of the Os-akt1 mutant was significantly reduced
in both root and shoot compared with wild-type plants (Figure
3H). Since Os-AKT1 is an inward K+ channel, this K+ deficiency
in Os-akt1 might be due to the defect of Os-AKT1-mediated K+
uptake. The results of K+ depletion experiments (Drew et al.,
1984) showed that the K+ uptake in Os-akt1 was obviously
slower than that in the wild type (Figure 3I). These results
demonstrated that the loss of function of Os-AKT1 led to the
reduction of K+ uptake in Os-akt1 mutants, which caused
growth inhibition and K+-deficient symptoms in mutant plants.
The function of Os-AKT1 was further confirmed using two
complementation lines for the Os-akt1 mutant. As shown in
Figure 4, the expression of Os-AKT1 in these two transgenic
lines was recovered and the growth inhibition and K+-deficient
symptoms were relieved.
The growth of Os-akt1 mutant plants was inhibited throughout
development (Figure 5). The heading and grain-filling stages
were delayed in Os-akt1 (Figures 5A and 5B). In addition, the
lesion of Os-AKT1 also impaired grain yield. The grain number,
seed set percentage, and 100-grain weight of the main panicle
were all significantly reduced in Os-akt1 (Figures 5C to 5G).
Electrophysiological Analysis of Rice Root Cell Protoplasts
To analyze the Os-AKT1-mediated K+ currents in rice, we tested
the whole-cell inward K+ currents in root cell protoplasts isolated
from Os-akt1 mutant and wild-type seedlings using patchclamping methods. In wild-type plants, ;37% of the recorded
root cell protoplasts showed inward K+ currents, but this proportion was significantly reduced in the Os-akt1 mutant (Table
1). Furthermore, the magnitude of inward K+ currents recorded
in Os-akt1 mutant root cells was much smaller than that in wildtype cells, at only 14% of the currents recorded from wild-type
plant cells at 2180 mV (Figure 6, Table 1). In the Os-akt1
complementation line, the magnitude of inward K+ currents was
completely recovered (Supplemental Figure 4) even though the
inward K+ currents could be recorded only in some of the root
cell protoplasts (34%). Although the K+ currents in Os-akt1 were
reduced, the voltage dependence of the K+ currents was not
altered (Figure 6C, Table 1), indicating that the K+ currents in
both Os-akt1 and wild-type root cells were derived from OsAKT1. We conclude that lesion of Os-AKT1 decreased the inward K+ currents in root cells, which led to the reduction of K+
uptake in Os-akt1 mutant roots.
Identification of Os-AKT1 Regulators
The previous reports have revealed that the activation of AKT1
channel in Arabidopsis root cells requires protein kinase CIPK23
and calcium sensor CBL1/9 (Li et al., 2006; Xu et al., 2006). Since
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Figure 2. Functional Characterization of Os-AKT1 in Yeast and Arabidopsis.
(A) Os-AKT1 and AKT1 complement the K+ uptake-deficient yeast mutant R5421 on AP medium containing different K+ concentrations. The yeast strain
R757 was used as a positive control. Three independent experiments were performed.
(B) The K+ uptake kinetic analysis of Os-AKT1 and AKT1 in yeast. The data points are shown as means 6 SE (n = 3).
(C) Phenotype comparison of wild-type Arabidopsis (Col), akt1 mutant, and two transgenic lines (akt1/Os-AKT1-1 and akt1/Os-AKT1-2) grown on MS
and LK (100 mM K+) medium for 7 d.
(D) Real-time PCR verification of Os-AKT1 and AKT1 expression in different plant materials.
(E) Comparison of K+ content in different plant materials. The K+ content of roots and shoots was determined after the plants grown on MS and LK
medium for 7 d. Data are shown as means 6 SE (n = 3). Student’s t test (*P < 0.05 and **P < 0.01) was used to analyze statistical significance.
(F) Patch-clamp whole-cell recordings of inward K+ currents in Arabidopsis root cell protoplasts. The plant materials are indicated above each recording. The voltage protocols, as well as time and current scale bars for the recordings, are shown inside the figure.
(G) The I-V (current-voltage) relationship of the steady state whole-cell inward K+ currents in root cell protoplasts. The data are derived from the
recordings as shown in (F) and presented as means 6 SE (Col, n = 35; akt1, n = 6; akt1/Os-AKT1-2, n = 28).
(H) The voltage dependence of inward K+ currents in root cell protoplasts isolated from different plants. The solid lines represented the best fits
according to the Boltzmann function: G/Gmax = 1/(1+exp((Vm2V1/2)/S)). G (conductance) was calculated as G=I/(Vm2EK), where I is the steady state
current at voltage Vm. V1/2 is the membrane potential at which the conductance is half-maximal and S is a slope factor. The data are derived from the
recordings as shown in (F) and presented as means 6 SE (Col, n = 35; akt1, n = 6; akt1/Os-AKT1-2, n = 28).
Os-AKT1 Functions in Rice K+ Uptake
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Figure 3. Phenotype of Rice Os-akt1 Mutant.
(A) The structure of the Os-AKT1 gene. The black boxes indicate exons and the lines represent introns. The T-DNA insertion site in the Os-akt1 mutant
is shown using an arrow.
(B) Real-time PCR verification of Os-AKT1 expression in Dongjin and Os-akt1 mutant. The data are presented as means 6 SE (n = 3).
(C) DNA gel blot analysis of T-DNA insertion in Os-akt1. Lanes 1, 4, and 7 were Dongjin (negative control); lanes 2, 5, and 8 were Os-akt1 mutant; and
lanes 3, 6, and 9 were the plasmid containing the HPT gene (positive control). Genomic DNA was isolated from rice leaves and digested using BamHI
(left), SacI (middle), or HindIII (right). A DNA fragment of HPT included in the T-DNA insertion fragment was used as probe.
(D) and (E) Phenotype comparison between Dongjin and Os-akt1 mutant. The photographs of whole seedlings (D) and first leaves (E) were taken after
the rice seedlings were grown in hydroponic solution for 7 d. Bars in (D) = 15 cm; bars in (E) = 2 cm.
(F) to (H) Seedling length (F), dry weight (G), and K+ content (H) of Dongjin and Os-akt1 mutant after growth in hydroponic solution for 7 d. Data are
shown as means 6 SE (n = 3). Student’s t test (*P < 0.05 and **P < 0.01) was used to analyze statistical significance of differences between genotypes.
(I) Comparison of K+ uptake ability between Dongjin and Os-akt1 using the K+ depletion method. Data are shown as means 6 SE (n = 3).
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Figure 4. Phenotype of Rice Os-akt1 Complementation Plants.
(A) Phenotype comparison of different plant materials. The photographs of whole seedlings and first leaves were taken after the rice seedlings were
grown in hydroponic solution for 7 d. The seedlings transformed with empty vector pBI121 were used as controls. COM1 and COM2 represent the two
complementation lines of Os-akt1 mutant. Bars = 5 cm.
(B) Real-time PCR verification of Os-AKT1 expression in different plant materials. The data are presented as means 6 SE (n = 3).
(C) and (D) Dry weight (C) and K+ content (D) of different plant materials after the rice seedlings were grown in hydroponic solution for 7 d. Data
are shown as means 6 SE (n = 3). Student’s t test (*P < 0.05 and **P < 0.01) was used to analyze statistical significance of differences from the
wild type.
Os-AKT1 and Arabidopsis AKT1 displayed the similar K+ transport
activity (Figures 2A and 2B) and Os-AKT1 could also mediate inward K+ currents in Arabidopsis root cells (Figures 2F and 2G), we
further hypothesized that Os-AKT1 may be also regulated by OsCIPKs and Os-CBLs in rice.
Based on sequence alignment, we cloned the Os-CIPK23 and
Os-CBL1 genes from rice that showed highest similarities with
Arabidopsis CIPK23 and CBL1, respectively. Then, we assayed
the protein interaction among Os-AKT1, Os-CIPK23, and OsCBL1. Yeast two-hybrid assays showed that Os-CIPK23 could
interact with Os-CBL1, as well as the cytosolic region of OsAKT1 (Os-AKT1-C, from 334 to 935 amino acids) (Figure 7A).
This interaction was further validated by bimolecular fluorescence
complementation (BiFC) assays in tobacco leaves. The yellow
fluorescent protein (YFP) fluorescence was detected at the PM in
tobacco epidermis in which Os-CIPK23-YN and Os-CBL1-YC
were coexpressed, indicating interaction between Os-CIPK23
and Os-CBL1 (Figure 7B). We did not detect interaction fluorescence in leaves coexpressing Os-AKT1-YC and Os-CIPK23-YN
(Figure 7B). However, when Os-CBL1 was coexpressed with OsAKT1-YC and Os-CIPK23-YN, the YFP fluorescence could be
observed at the PM in epidermis (Figure 7B). These results suggested that the interaction of Os-CIPK23 and Os-AKT1 in plant
cells requires the presence of Os-CBL1. Os-CBL1 may recruit OsCIPK23 to the PM, so that Os-CIPK23 can interact with PMlocated Os-AKT1.
Os-AKT1 Functions in Rice K+ Uptake
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Figure 5. Lesion of Os-AKT1 Inhibits Plant Growth and Impairs Grain Yield.
(A) and (B) Phenotype comparison of Dongjin and Os-akt1 mutant plants at tillering stage (A) and grain-filling stage (B). Bar in (A) = 10 cm; bar in (B) =
15 cm.
(C) Comparison of main panicle between Dongjin and Os-akt1 mutant plants. Bar = 5 cm.
(D) to (G) Comparison of main panicle length (D), grain number (E), seed set percentage (F), and 100-grain weight (G) between Dongjin and Os-akt1
mutant plants. Data are shown as means 6 SE (n = 10). Student’s t test (*P < 0.05 and **P < 0.01) was used to analyze statistical significance.
Activation of Os-AKT1 by Os-CBL1 and Os-CIPK23
Based on the protein interaction results as shown in Figures 7A
and 7B, we hypothesized that the activity of Os-AKT1 may be
regulated by Os-CIPK23 and Os-CBL1. Thus, we tested for OsCBL1-Os-CIPK23 regulation of Os-AKT1 in Xenopus laevis oocytes using a similar strategy as described in a previous report
(Xu et al., 2006). In X. laevis oocytes, neither Arabidopsis AKT1
nor Os-AKT1 alone could form functional K+ channel. However,
Arabidopsis AKT1 could be activated by both Arabidopsis
CBL1-CIPK23 complex and rice CBL1-CIPK23 complex. By
contrast, neither rice CBL1-CIPK23 nor Arabidopsis CBL1CIPK23 could activate Os-AKT1 in oocytes (Supplemental
Figure 5), suggesting differences between these two channels.
Os-AKT1 may require some other regulators besides rice CBLCIPK complex.
A previous report showed that Os-AKT1 alone could mediate
inward K+ currents in HEK293 cells (Fuchs et al., 2005). We
further tested the regulation of Os-CIPK23 and Os-CBL1 in
HEK293 cells. When Os-CBL1-GFP (green fluorescent protein)
and Os-CIPK23 were cotransfected with Os-AKT1 into HEK293
cells, the whole-cell inward K+ currents were remarkably increased compared with the cells transfected with Os-AKT1
alone (Figures 7C and 7D). After the cotransfection with OsCBL1-GFP and Os-CIPK23, the current density of Os-AKT1mediated inward K+ currents was increased from 2365 6 30 pA/pF
to 21094 6 132 pA/pF at 2200 mV (Figure 7D). However, the
voltage dependence of Os-AKT1 was not affected (V1/2 Os-AKT1 =
2188 6 4 mV, V1/2 Os-CBL1+Os-CIPK23+Os-AKT1 = 2184 6 2 mV; Figure 7E).
We also found that Os-CIPK23-GFP alone could not enhance OsAKT1 activity (Figures 7C and 7D), which indicated that the activation of Os-AKT1 required the presence of both Os-CBL1 and
Os-CIPK23. In this experiment, the GFP tag fused to Os-CBL1
or Os-CIPK23 was used as a reporter for transfection efficiency.
In addition, we also searched for any other Os-CIPKs that
activate Os-AKT1. We cloned 25 rice CIPK genes and tested for
interaction between these Os-CIPK proteins and Os-AKT1 using
yeast two-hybrid assays and BiFC experiments. Besides OsCIPK23, there were two Os-CIPK proteins (Os-CIPK3 and 19)
interacting with Os-AKT1 (Supplemental Figure 6). We further
tested if these two Os-CIPKs could activate Os-AKT1 in
HEK293 cells. As shown in Supplemental Figure 7, only OsCIPK19 could enhance Os-AKT1 inward K+ currents in the
presence of Os-CBL1. However, the activation mediated by OsCIPK19 was much weaker than that mediated by Os-CIPK23.
Since Os-AKT1 is regulated by CBL proteins, it is possible
that Ca2+ signaling might be involved in the regulation of OsAKT1. We tested for Ca2+-dependent activation of Os-AKT1 in
HEK293 cells. The results showed that activation of Os-AKT1
alone was dependent on cytosolic Ca2+ concentration. A high
cytosolic Ca 2+ concentration could enhance the Os-AKT1
activity (Supplemental Figure 8). After coexpression with rice
CBL1-CIPK23, the activation of Os-AKT1 was still dependent on
the cytosolic Ca2+ concentration (Supplemental Figure 8). In
addition, an Os-CBL1 EF-hand mutation (E172Q in the fourth
EF-hand) was used to test the Ca2+-dependent activation of OsAKT1. In HEK293 cells, the rice CBL1(E172Q)-CIPK23 could no
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Table 1. Electrophysiological Analysis of Rice Root Cell Protoplasts in Patch-Clamp Whole-Cell Recording Experiments
Rice Seedlings
No. of Recorded Cells
No. of Cells with Currents
Current Density at 2180 mV (pA/pF)
Normalized Gmax (nS/pF)
V1/2 (mV)
Dongjin
Os-akt1
144
136
53 (36.8%)
28 (20.6%)
2206 6 12
229 6 3
2.44 6 0.86
0.25 6 0.23
2162 6 6
2167 6 5
Data are shown as means 6
SE.
longer activate Os-AKT1 (Supplemental Figure 8). These data
indicated that the regulation of Os-AKT1 activity may be dependent on cytosolic Ca2+ signaling, mediated by Os-CBL1.
Physiological Function Analysis of Os-CIPK23 and Os-CBL1
To further characterize the functions of Os-CIPK23 and OsCBL1, Arabidopsis lks1 mutant (a CIPK23 knockout mutant, also
named the cipk23 mutant; Xu et al., 2006) and cbl1 cbl9 double
mutant plants (Xu et al., 2006) were transformed with Os-CIPK23
and Os-CBL1, respectively. The lks1 and cbl1 cbl9 mutant
plants displayed the similar sensitive phenotype as akt1 under
low K+ conditions (Xu et al., 2006). The Os-CIPK23 and OsCBL1 could rescue the sensitive phenotype of lks1 and cbl1
cbl9 in the transgenic lines (lks1/Os-CIPK23 and cbl1 cbl9/OsCBL1) (Figures 7F and 7G). Furthermore, the K+ contents in
these transgenic lines were also resumed (Supplemental Figure 9).
To further characterize the function of Os-CIPK23 in rice, we
tested the phenotype of Os-CIPK23 RNA interference (RNAi)
lines (Yang et al., 2008; O. sativa ssp japonica cv Nipponbare).
Similar to Os-akt1 mutant, the two Os-CIPK23 RNAi lines both
displayed the K+ deficiency symptoms, growth inhibition, and
leaf brown spots, especially under low K+ conditions (Figures 8A
and 8B). The expression of Os-CIPK23 in these two RNAi lines
was detected using real-time PCR assays (Figure 8C). The shoot
length and dry weight was reduced in the RNAi lines (Figures 8D
and 8E). The decline of K+ content in the RNAi lines under low K+
conditions (Figure 8F) was consistent with the K+ deficiency
symptoms in Figures 8A and 8B. The K+ depletion experiments
indicated that the K+ deficiency in Os-CIPK23 RNAi lines may
result from the reduction of K+ uptake capacity (Figure 8G).
These data demonstrated that Os-CIPK23 plays important role
in rice K+ uptake.
For the Os-cbl1 mutant, the mutant plants did not show a remarkable low-K+-sensitive phenotype compared with wild-type
seedlings even though the K+ content in Os-cbl1 mutant roots
was reduced (Supplemental Figure 10). The K+ uptake capacity of
the Os-cbl1 mutant was slightly reduced (Supplemental Figure 10F),
which suggested Os-CBL1 may be partially involved in K+ uptake
in rice roots.
DISCUSSION
The Role of Os-AKT1 in Rice K+ Uptake
In Arabidopsis, AKT1 mediates root K+ uptake over a wide range
of external K+ concentrations (Lagarde et al., 1996; Hirsch et al.,
1998). Here, we report that Os-AKT1, an AKT1 homolog, conducts the K+ uptake in rice roots. The GUS activity analysis
indicated that Os-AKT1 is abundantly expressed in rice roots
(Figure 1B), especially in root hairs and epidermis (Figures 1C
and 1D), which is the precondition that Os-AKT1 mediates the
K+ uptake from the soils. The K+ uptake test in both yeast and
Arabidopsis (Figures 2A and 2C) indicated that Os-AKT1 could
mediate the K+ uptake similar to the Arabidopsis AKT1 channel
(Figure 2B).
The Os-akt1 mutant plants showed a typical low-K+-sensitive
phenotype compared with the wild-type plants (Figures 3D and
3E). The K+ content in Os-akt1 mutant was significantly reduced
(Figure 3H), suggesting that the low-K+-sensitive phenotype of
Os-akt1 may be due to the reduction of K+ uptake. This is
consistent with the results from patch-clamping experiments
and K+ depletion assays, in which the inward K+ fluxes and
K+ uptake rate in Os-akt1 mutant root cells were significantly
Figure 6. Electrophysiological Analysis of Inward K+ Currents in Rice
Root Cells.
(A) Patch-clamp whole-cell recordings of inward K+ currents in rice root
cell protoplasts isolated from Dongjin and Os-akt1 mutant. The voltage
protocols, as well as time and current scale bars for the recordings, are
shown.
(B) The I-V (current-voltage) relationship of the steady state whole-cell
inward K+ currents in rice root cell protoplasts. The data are derived from
the recordings as shown in (A) and presented as means 6 SE (Dongjin,
n = 43; Os-akt1, n = 28).
(C) The G-V (conductance-voltage) relationship of the steady state
whole-cell inward K+ currents in rice root cell protoplasts. The solid lines
represented the best fits of conductance (G) according to the Boltzmann
function. The data are derived from the recordings as shown in (A) and
presented as means 6 SE (Dongjin, n = 43; Os-akt1, n = 28).
Os-AKT1 Functions in Rice K+ Uptake
Figure 7. Os-CBL1 and Os-CIPK23 Enhance Os-AKT1 Activity.
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The Plant Cell
reduced compared with those of wild-type rice (Figures 3I and
6A). Since K+ is a key nutrient factor that influences plant
growth, development, and crop yield, it is plausible to propose
that the reduction of K+ uptake in the Os-akt1 mutant may result
in the inhibition of plant growth and the decline of grain yield. We
indeed observed growth inhibition and development delay in
Os-akt1 mutant plants (Figures 5A and 5B), and the grain yield
was also impaired in the mutant plants (Figures 5C to 5G).
Characteristic Comparison of Rice and Arabidopsis AKT1
The yeast complementation experiments showed that rice and
Arabidopsis AKT1 not only rescued the growth defect of R5421
under low K+ conditions (<250 mM K+), but also enhanced the
R5421 growth under higher K+ conditions (1 and 5 mM K+)
compared with the wild-type strain R757 (Figure 2A). In addition,
the electrophysiological experiments in HEK293 cells also indicated that Os-AKT1 could mediate inward K+ currents even
though the external K+ concentrations varied from 10 mM to
1 mM (Supplemental Figure 11). This suggests that similar to
Arabidopsis AKT1, Os-AKT1 could also mediate K+ uptake over
a wide range of external K+ concentrations.
Os-AKT1 could rescue the low-K+-sensitive phenotype of the
Arabidopsis akt1 mutant (Figure 2C) because the inward K+
currents were recovered by Os-AKT1 in akt1/Os-AKT1 root cells
(Figure 2F). However, Os-AKT1-mediated K+ currents were obviously different from the currents in Arabidopsis wild-type
plants (Figures 2F and 2G), suggesting that there are different
properties for these two channels. The activation curve analysis
derived from patch-clamp recordings showed that the halfactivation voltage of Os-AKT1 (V1/2 = 2173 6 5 mV) was much
more negative than that of AKT1 (V1/2 = 2133 6 4 mV) (Figure
2H), which suggests that the activation threshold of Os-AKT1 is
much more negative than AKT1. In addition, we determined the
pH sensitivity of these two channels. Along with the increment of
extracellular pH from pH 5.0 to 6.5, the V1/2 of AKT1 channel (in
Col plants) and Os-AKT1 channel (in akt1/Os-AKT1 transgenic
plants) were both slightly shifted in the negative direction
(Supplemental Figure 12). However, the channel activity and V1/2
of AKT1 were more sensitive to extracellular pH compared with
Os-AKT1 (Supplemental Figure 12).
The previous reports showed that, in X. laevis oocytes, outward K+ currents (K+ leakage) were recorded in AKT1-expressed
oocytes under low external K+ concentrations, when the test
voltage was between the K+ reversal potential and the AKT1
activation threshold (Duby et al., 2008; Geiger et al., 2009).
However, At-KC1 channel subunit could interact with AKT1 and
restrain AKT1-mediated K+ leakage under low external K+ concentrations by shifting the voltage dependence of AKT1 in the
negative direction (Duby et al., 2008; Geiger et al., 2009; Wang
et al., 2010). In Arabidopsis, KC1 is a general modulator of
inward Shaker K+ channels that negatively shifts the channel
activation threshold and limits the K+ leakage under low K+
conditions (Jeanguenin et al., 2011). A homolog of At-KC1 was
not found in the rice genome (Fuchs et al., 2005). It is therefore
probable that Os-AKT1 is not regulated by a KC1-like channel in
rice. However, it was observed that the half activation voltage of
Os-AKT1 was more negative in akt1/Os-AKT1 Arabidopsis root
cells (V1/2 = 2173 6 5 mV) than in rice root cells (V1/2 = 2162 6
6 mV). We speculate that At-KC1 may also regulate Os-AKT1 in
akt1/Os-AKT1 Arabidopsis lines and negatively shift the activation potential of Os-AKT1. The interaction between Os-AKT1
and At-KC1 was also tested, and At-KC1 indeed interacted with
Os-AKT1 at the PM (Supplemental Figure 13).
Despite the absence of a KC1-like channel in rice, it seems
that Os-AKT1 alone could restrain the K+ leakage under low K+
conditions. In HEK293 cells, outward K+ currents (K+ leakage)
were not observed in cells transfected with Os-AKT1, even
when the external K+ concentration was reduced from 1 mM to
10 mM (Supplemental Figure 11). The results of patch-clamping
experiments from root cells showed that the voltage dependence of Os-AKT1 (V1/2 = 2162 6 6 mV in rice root cell
protoplasts; Figure 6C) was much more negative than that of
AKT1 (V1/2 = 2133 6 4 mV in Arabidopsis root cell protoplasts;
Figure 2H). That may be the reason why Os-AKT1 does not
require a KC1-like channel and can restrain the K+ leakage alone
under low K+ conditions.
The patch-clamping experiments indicated that the Os-AKT1mediated inward K+ currents could be recorded only in a portion
of the rice root cell protoplasts (Table 1), which is not consistent
with the ubiquitous expression of Os-AKT1 in rice root cells
(Figures 1C and 1D). This might suggest that there is differential
regulation of Os-AKT1 in individual root cell types, so that the
Os-AKT1 activity is fine-tuned in the specific cell types.
The previous data indicated that AKT1 may not rely on the
transcriptional regulation. In Arabidopsis, K+ deprivation did not
Figure 7. (continued).
(A) Yeast two-hybrid analysis of Os-CIPK23 interaction with Os-CBL1 and the cytosolic region of Os-AKT1 (Os-AKT1-C).
(B) BiFC assays of Os-CIPK23 interaction with Os-CBL1 and Os-AKT1 in N. benthamiana leaves. Bar = 100 mm.
(C) Patch-clamp whole-cell recordings of inward K+ currents in HEK293 cells expressing different combinations of Os-CBL1, Os-CIPK23, and OsAKT1. The voltage protocols, as well as time and current scale bars for the recordings, are shown.
(D) The I-V relationship of the steady state whole-cell inward K+ currents in HEK293 cells. The data are derived from the recordings as shown in (C) and
presented as means 6 SE (Os-AKT1, n = 59; Os-CBL1-GFP+Os-CIPK23, n = 20; Os-CIPK23-GFP+Os-AKT1, n = 8; Os-CBL1-GFP+Os-CIPK23+OsAKT1, n = 52).
(E) The G-V relationship of the steady state whole-cell inward K+ currents in HEK293 cells. The solid lines represented the best fits of conductance (G)
according to the Boltzmann function. The data are derived from the recordings as shown in (C) and presented as means 6 SE (Os-AKT1, n = 59; OsCIPK23-GFP+Os-AKT1, n = 8; Os-CBL1-GFP+Os-CIPK23+Os-AKT1, n = 52).
(F) and (G) Phenotype of Arabidopsis transgenic lines expressing Os-CIPK23 (F) and Os-CBL1 (G) in the lks1 and cbl1 cbl9 mutant backgrounds,
respectively. The photographs were taken after the plants were grown on MS and LK medium for 7 d.
Os-AKT1 Functions in Rice K+ Uptake
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Figure 8. Phenotype of Os-CIPK23 RNAi Plants.
(A) and (B) Phenotype comparison between Nipponbare (Np) and Os-CIPK23 RNAi plants (23RNAi-1 and 23RNAi-2). The photographs of whole
seedlings (A) and first leaves (B) were taken after the rice seedlings were grown in hydroponic solution for 7 d. Bars in (A) = 15 cm; bars in (B) = 2 cm.
(C) Real-time PCR verification of Os-CIPK23 expression in Nipponbare and Os-CIPK23 RNAi plants.
(D) to (F) Seedling length (D), dry weight (E), and K+ content (F) of Nipponbare and Os-CIPK23 RNAi plants after growth in hydroponic solution for 7 d.
Data are shown as means 6 SE (n = 3). Student’s t test (*P < 0.05 and **P < 0.01) was used to analyze statistical significance of difference from the wild
type.
(G) Comparison of K+ uptake ability between Nipponbare and Os-CIPK23 RNAi plants using the K+ depletion method. Data are shown as means 6 SE
(n = 3).
affect the expression of AKT1 (Kim et al., 1998; Maathuis et al.,
2003; Pilot et al., 2003; Ahn et al., 2004; Hampton et al., 2004;
Gierth et al., 2005). Arabidopsis AKT1-overexpressing lines do
not show enhanced low-K+-tolerance phenotypes nor significantly increased inward K+ currents in root cells (Xu et al., 2006).
These results suggest that the regulation of Arabidopsis AKT1
may occur at posttranslational level (Wang et al., 2010). As for
Os-AKT1, the real-time PCR results and GUS assays also
confirmed that the expression of Os-AKT1 in rice roots was not
changed under K+ deficiency conditions (Supplemental Figure 3),
indicating that Os-AKT1 may be also regulated at posttranslational
level.
The Regulatory Mechanism of AKT1-Like Channels in Plants
In Arabidopsis, the activity of AKT1 channel is controlled by
CBL1/9 and CIPK23 (Li et al., 2006; Xu et al., 2006). Similar
regulatory mechanisms involving CBL-CIPK complexes modulating K+ channel were reported for several different plant species based on electrophysiological analyses. Two AKT1-like
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channels, Hv-AKT1 from barley and Vv-K1.1 from grapevine
both could be activated by Arabidopsis CBL1 and CIPK23 in
X. laevis oocytes (Boscari et al., 2009; Cuéllar et al., 2010).
Furthermore, another K+ channel from grapevine, Vv-K1.2, was
characterized as a voltage-gated inward K+ channel. In X. laevis
oocytes, Vv-K1.2 alone could not form functional channel. However, after coexpression with Vv-CBL01-Vv-CIPK04 or Vv-CBL02Vv-CIPK03, Vv-K1.2 was capable of mediating inward K+ currents
(Cuéllar et al., 2013). It seems that the regulation of AKT1-like
channels via a CBL-CIPK complex might be a universal mechanism in different plant species.
In this study, we tried to identify the candidate Os-CBLs and
Os-CIPKs that may regulate Os-AKT1 activity. Based on the
sequence alignment between rice and Arabidopsis, we cloned
Os-CBL1 and Os-CIPK23 from rice. In HEK293 cells, Os-AKT1
alone could mediate the inward K+ currents (Fuchs et al., 2005),
whose activity was further enhanced when coexpressed with
rice or Arabidopsis CBL1-CIPK23 (Figures 7C and 7D; Supplemental
Figure 14). Os-CBL1 and Os-CIPK23 could rescue the lowK+-sensitive phenotype of Arabidopsis cbl1 cbl9 and lks1 mutants,
respectively (Figures 7F and 7G). These results indicated that the
function of CBL and CIPK proteins in K+ channel regulation may be
conserved in different plant species.
The phenotype test showed that Os-CIPK23 RNAi lines exhibited similar K+-deficient symptoms as the Os-akt1 mutant
under low K+ conditions (Figures 3E and 8B). In these two mutant lines, the K+ uptake rates were reduced (Figures 3I and 8G),
which led to the decline of K+ content (Figures 3H and 8F) and
inhibition of plant growth (Figures 3G and 8E). Therefore, the OsCIPK23 should be the functional regulator of Os-AKT1 in rice. As
for Os-CBL1, the Os-cbl1 mutant did not show an obvious lowK+-sensitive phenotype (Supplemental Figure 10). There are two
possible reasons for this. On the one hand, the Os-cbl1 mutant
used in this study is a knockdown line (Supplemental Figure 10).
The leaky expression of Os-CBL1 might be sufficient to accomplish its physiological function in Os-AKT1 regulation. On
the other hand, there might be some other CBLs in rice that
are functionally redundant with Os-CBL1. This would be quite
similar to the situation in Arabidopsis, in which only the cbl1 cbl9
double mutant showed obvious low-K+-sensitive phenotypes
and neither cbl1 nor cbl9 single mutants exhibited the sensitive
phenotype (Xu et al., 2006).
In conclusion, our findings demonstrate that the inward K+
channel Os-AKT1 functions in K+ uptake in rice roots, whose
activity is regulated by Os-CBL1 and Os-CIPK23. These findings
may provide insight to improve rice K+ uptake efficiency and
enhance rice tolerance to K+ deficiency stress.
temperature for one night. Then, the seeds were sown on wet filter papers.
Uniformly germinated seeds were selected and transplanted into nutrient
solution one week later. The 14-d-old seedlings were transferred into
nutrient solution with different K+ concentrations (1 mM and 100 mM). The
nutrient solution (pH 5.7) consisted of macronutrients (mg L21): NH4NO3
114.25, NaH2PO4$2H2O 50.38, K2SO4 89.25, CaCl2 110.75, MgSO4$7H2O
405, FeSO4$7H2O 34.75, Na2EDTA 46.53; and micronutrients (mg L21):
MnCl2$4H2O 1.88, (NH4)6Mo7O24$4H2O 0.09, H3BO3 1.17, ZnSO4$7H2O
0.44, CuSO4$5H2O 0.39 (International Rice Research Institute, Philippines). The nutrient solution was renewed every 2 d. For the low K+ (100
mM) hydroponics, the amount of K2SO4 was reduced to achieve the low K+
concentration. Other components in nutrient solution were not changed.
The Arabidopsis thaliana seeds were germinated on Murashige and
Skoog (MS) medium containing 0.8% (w/v) agar and 3% sucrose at 22°C
under constant illumination. Then, 4-d-old seedlings were transferred to
MS and low-K+ (LK; 100 mM) medium, which was described previously (Xu
et al., 2006). The low-K+ phenotype was observed after 7 d.
Subcellular Localization Analysis
The coding sequence of Os-AKT1 was fused with GFP in the pCAMBIA1300 vector. The plasmids pCAMBIA1300-Os-AKT1-GFP and pGPTVII et al., 2008) were electroporated into Agrobacterium
CBL1n-OFP (Batistic
tumefaciens (GV3101) using a Bio-Rad Gene Pulser. The methods for
plasmid transformation and expression were described in the previous
literature (Rajamäki and Valkonen, 2009).
GUS Assays
The 1010-bp fragment before the ATG codon of Os-AKT1 was cloned
from the japonica rice cultivar Nipponbare. This fragment was used as OsAKT1 promoter in this study and constructed into pCAMBIA1381 vector.
The plasmid was transformed into Nipponbare rice via callus transformation, which was mediated by Agrobacterium (EHA105). The T2
heterozygous transgenic rice plants were treated with the GUS staining
solution which contained 1% (v/v) N,N-dimethyl formamide, 0.1% X-Gluc,
0.1% Triton X-100, 0.1 M PBS, 0.05 mM K3Fe(CN)6, and 0.05 mM K4Fe
(CN)6$3H2O. Then, the rice plants were immersed in 75% (v/v) ethanol.
The GUS-stained roots and leaves were sliced into semithin sections for
microscopy observation.
K+ Content Analysis
METHODS
The 14-d-old rice seedlings were transferred into nutrient solution with
different K+ concentrations (1 mM and 100 mM) and treated for 7 d. The
shoots and roots were collected, rinsed with deionized water, and dried at
80°C to constant weight in paper bags. The dry weights of samples were
measured as dry biomass. Then the dry plant tissues were incinerated in
a muffle furnace at 575°C for 9 h. The ashes were dissolved with 0.1 N
hydrochloric acid for 4 h and then diluted with 0.1 N hydrochloric acid into
different multiples. The K+ concentrations were determined by Zeeman
atomic absorption spectrophotometry (Hitachi Z-2000). The method of
K + content analysis for Arabidopsis plants was described previously
(Xu et al., 2006).
Plant Material and Growth Conditions
Real-Time PCR
For rice (Oryza sativa) harvest, the rice seeds were first germinated in Petri
plates. One week later, the rice seedlings were transplanted into growing trays
with soil mixture (rich soil: vermiculite = 1:1, v/v) and kept in growth chamber
with a 12-h-light (28°C)/12-h-dark (24°C) photoperiod. About 3 weeks later, the
plants were transplanted into the field and grown until harvest.
The rice seeds were sterilized with 20% NaClO for 1 h, soaked in
deionized water at room temperature for 2 d, and pregerminated at room
The total RNA was extracted from rice seedlings grown under normal
conditions using TRIzol reagent (Invitrogen). The 4 mg-DNase-treated
total RNA was reverse transcribed into cDNA with random primers. The
cDNA was diluted 40 times, and 6 mL diluted cDNA was used as the
template in each well for quantitative real-time PCR analysis. The cDNA
was amplified using Power SYBR Green PCR Master Mix (Applied Biosystems) on the ABI 7500 thermocycler (Applied Biosystems). The
Os-AKT1 Functions in Rice K+ Uptake
amplification reactions were performed in a total volume of 20 mL, which
contained 6 mL cDNA, 2 mL forward and reverse primers (1 mM), 10 mL
SYBR Green premix, and 2 mL PCR-grade sterile water. The PCR was
programmed as follows: 95°C for 10 min, followed by 40 cycles of 95°C
for 15 s and 60°C for 1 min. An Actin gene was used as an internal
standard to normalize the expression data for the tested gene. All the
primers used in this experiment are listed in Supplemental Table 1.
Three replications were performed for each sample. The fluorescence
signal was detected during the annealing step. All experiments were repeated three times.
Yeast Complementation
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Generation of Arabidopsis Transgenic Plants
Full-length coding sequences of Os-AKT1, Os-CIPK23, and Os-CBL1
were constructed into the overexpression vector pUN1301 (vector
pCAMBIA1301 modified with ubiquitin promoter instead of cauliflower
mosaic virus 35S promoter; Yu et al., 2007). The three constructs were
transformed into akt1, lks1, and cbl1 cbl9 Arabidopsis mutant plants,
respectively. The Arabidopsis transformation was performed using the
floral dip method with Agrobacterium (strain GV3101; Clough and Bent,
1998). The T4 homozygous transgenic plants were used to examine the
phenotype under low-K+ conditions. The expression of Os-AKT1 in transgenic plants was detected using real-time PCR. The Os-CIPK23 and
Os-CBL1 expression in transgenic plants were detected using RT-PCR.
The coding sequences of Os-AKT1 and AKT1 were constructed into
p416-GPD vector and transformed into the yeast strain R5421 (trk1D
trk2D), in which the two endogenous K+ transporter genes (TRK1, 2) were
deleted. The yeast strain R757 was used as a positive control. Single
colonies were picked from different transformed yeast cells and cultured
at 30°C overnight in 2 mL YPDA medium containing 100 mM KCl until
the OD600 reached 0.8. The yeast cells were collected by centrifugation
at 6000g for 1 min and washed in double-distilled water three times.
Cells were resuspended in double-distilled water with the OD600 0.8,
and 10-fold serial diluted cultures were incubated on AP plates containing different K + concentrations. These plates were incubated at
30°C for 2 d.
For kinetic analysis of K+ uptake in yeast, yeast colonies expressing
Os-AKT1 and AKT1 were cultured at 30°C overnight in 50 mL YPDA
medium, until the OD600 reached 2.5. The yeast cells were collected and
washed in double-distilled water for three times. Cells were resuspended
in double-distilled water with the OD600 3.0. The 30 mL liquid AP medium
containing different KCl concentrations was added in each 50-mL flask.
Yeast cells (100 mL) were transferred into the AP medium, and flasks were
shaken at 30°C. The OD600 values of the yeast cells were recorded every
1.5 h after the OD600 reached 0.2. The slope for each K+ concentration was
calculated according to the linear regression of the growth curves during
the logarithmic growth phase. The curve in the graph was obtained by
applying nonlinear regression analysis using the Michaelis-Menten equation
(Horie et al., 2011).
Rice seeds were germinated on half-strength MS medium containing
0.2% (w/v) plant phytagel and 3% sucrose at 28°C under constant illumination. For K+-depletion experiments (Drew et al., 1984), 7-d-old
seedlings were pretreated in starvation solution (0.2 mM CaSO4 and 5 mM
MES, pH 5.75 adjusted with Tris) at 28°C for 18 h. Each sample included
seven seedlings, the fresh weight of which was near 0.8 g. The tests
began 5 min after transfer of seedlings into the depletion solution (0.25
mM KNO3, 0.2 mM CaSO4, and 5 mM MES, pH 5.75 adjusted with Tris).
The experiments were conducted at 28°C in the light, and all the samples
were shaking on a shaking table during the experiments (Xu et al., 2006;
modified). The solution samples were collected at different time points as
indicated, and the K+ concentrations were measured by Zeeman atomic
absorption spectrophotometry (Hitachi Z-2000).
Yeast Two-Hybrid Assays
DNA Gel Blot Analysis
The coding sequences of Os-CBL1 and the cytosolic region of Os-AKT1
(Os-AKT1-C, from N334) were cloned into pGADT7 vector. Os-CIPK23
coding sequence was introduced into pGBKT7 vector. The plasmids were
transformed into yeast strain AH109 for yeast two-hybrid assays following
the lithium acetate method (according to TRANFOR protocol). The positive
clones of Os-CBL1/Os-CIPK23 and Os-CIPK23/Os-AKT1-C were selected
from SC medium (-Leu-Trp, without 3-aminotriazole) incubated at 28°C after
3 d. Then, all of the positive clones were scribed on SC medium (-Leu-Trp)
as well as selective medium (-Leu-Trp-His-Ade) without 3-aminotriazole.
Four days later, b-galactosidase activity was detected, and photographs
were taken subsequently.
Genomic DNA was isolated from the leaves of Os-akt1 mutant and
Dongjin by the CTAB method. The 20 mg DNA was digested with different
restrictive enzymes, separated on 1% agarose (Invitrogen) gel, and
transferred onto Amersham Hybond-N + membrane (GE Healthcare).
Prehybridization, hybridization, and bolt washing were performed as
recommended by the manufacturer. A segment of the hygromycin resistance gene HPT (846 bp) was used as the probe and was labeled with
the digoxin labeling system (DIG DNA Labeling and Detection Kit; Roche).
The membrane was exposed to the Kodak XAR-50 x-ray film.
BiFC Assays
The coding regions of Os-CBL1 and Os-AKT1 were cloned into vector
pSPYCE (MR) (Waadt and Kudla, 2008; Waadt et al., 2008). The coding
sequence of Os-CIPK23 was cloned into vector pSPYNE (R) 173 (Waadt
and Kudla, 2008). The BiFC assays were performed as described
previously (Walter et al., 2004; Waadt and Kudla, 2008). The tested
construct pairs were expressed in leaves of Nicotiana benthamiana
for 5 d before microscopy observation. The YFP fluorescence in the
transformed leaves was imaged using a confocal laser scanning microscope (Leica DMIRE2).
Construction of Os-akt1 Complementation Rice Plants
The CDS of Os-AKT1 was cloned into the vector pBI121 (Chen et al.,
2003) with SmaI and XbaI. The 35S promoter in pBI121 vector was replaced by the Os-AKT1 promoter (1010 bp) using ScaI and SmaI. The
plasmid was transformed into the Os-akt1 mutant using a gene gun. The
empty vector pBI121 was also transformed into the Os-akt1 mutant, as
a control.
Kinetic Analysis of K+ Uptake
Protoplast Isolation and Patch-Clamp Whole-Cell Recording
The rice root cell protoplasts were isolated from 7-d-old primary roots of
rice seedlings that were grown on filter paper immersed in half-strength
MS with 1% sucrose at 25°C in the dark. The root tips (3 to 5 mm) were cut
into small pieces and incubated in enzyme solution at 28°C for 1 h to
release root cell protoplasts. The enzyme solution contained 1% (w/v)
cellulase (Onozuka R-10), 0.3% (w/v) pectolyase Y-23 (Onozuka), 0.5%
(w/v) BSA, 0.5% (w/v) polyvinylpyrrolidone, 1 mM CaCl2, 10 mM MES/Tris
(pH 5.6), and D-sorbitol (p = 300 mosmol kg21). The protoplasts were
filtered through 80-mm nylon mesh and washed three times with standard
solution, which contained 1 mM CaCl2, 10 mM MES/Tris (pH 5.6), and
D-sorbitol (p = 300 mosmol kg21). The protoplast suspension was kept on
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The Plant Cell
ice at least for 1 h and then used for patch-clamping experiments. Standard
whole-cell recording techniques were applied (Hamill et al., 1981). The bath
solution contained 100 mM K-gluconate, 10 mM CaCl2, 10 mM MES/Tris
(pH 5.6), and D-sorbitol (p = 300 mosmol kg21). The pipette solution
contained 150 mM K-gluconate, 2 mM MgCl2, 10 mM HEPES/Tris (pH 7.4),
and D-sorbitol (p = 300 mosmol kg21). The patch-clamping recordings were
conducted at ;20°C in dim light. Whole-cell currents were recorded using
an Axopatch 200B amplifier (Axon Instruments). The methods for Arabidopsis root cell protoplast isolation and patch-clamp whole-cell recording
were described previously (Xu et al., 2006).
Supplemental Figure 9. Os-CIPK23 and Os-CBL1 Rescue the K+
Content of Arabidopsis lks1 and cbl1 cbl9 Mutants.
Cell Culture, Transfection, and Whole-Cell Recording
Supplemental Figure 14. Electrophysiological Analysis of Os-AKT1
Regulation by Arabidopsis CBL1 and CIPK23 in HEK293 Cells.
For the patch-clamping recordings from HEK293 cells, we purchased
HEK293 cells from ATCC (American Type Culture Collection) and cultured
the cells in DMEM (Dulbecco’s modified eagle medium) with 4500 mg L21
glucose (Gibco) and 10% fetal calf serum (Gibco) for 24 h at 37°C, 5%
CO2. Then, the HEK293 cells were transfected with different combinations
of recombinant plasmids using Lipofectamine 2000 Transfection Reagent
(Invitrogen). Os-AKT1 was cloned into pcDNA3.1 vector (Invitrogen).
Os-CBL1 was cloned into pEGFP-N1 vector (Clontech). The vector
pBudCE4.1 (Invitrogen) was used for the simultaneous expression of both
Os-CBL1-GFP and Os-CIPKs. In pBudCE4.1 vector, Os-CBL1-GFP was
driven by CMV promoter, and Os-CIPKs were driven by EF-1a promoter.
The plasmids pcDNA3.1-Os-AKT1 and pBudCE4.1-Os-CBL1-GFP-OsCIPKs were cotransfected into HEK293 cells. Then, the transfected cells
were treated with Trypsin (Gibco), centrifuged at 160g for 5 min, and kept
on ice for patch-clamp recording. The cells with GFP fluorescence were
selected for whole-cell recording. The components of the pipette solution
were the same as described previously (Fuchs et al., 2005). The bath
solution contained 100 mM K-gluconate, 10 mM MES/Tris (pH 5.6), and
D-sorbitol (p = 350 mosmol kg21). The patch-clamp recordings were
conducted at ;20°C in dim light. Whole-cell currents were recorded using
an Axopatch 200B amplifier (Axon Instruments).
Accession Numbers
Sequence data from this article can be found in the Rice Genome Annotation Project under the following accession numbers: Os10g41510
for Os-CBL1, Os07g05620 for Os-CIPK23, Os03g20380 for Os-CIPK3,
Os05g43840 for Os-CIPK19, and Os01g45990 for Os-AKT1.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Amino Acid Sequence Alignment of Shaker
K+ Channels from Different Plant Species.
Supplemental Figure 2. Phylogenetic Analysis of AKT1-Like K+
Channels from Different Plant Species.
Supplemental Figure 3. Relative Expression of Os-AKT1 under LowK+ Stress.
Supplemental Figure 4. Electrophysiological Analysis of Inward K+
Currents in Rice Root Cells from Os-akt1 Complementation Line.
Supplemental Figure 5. Os-CBLs and Os-CIPKs Could Not Activate
Os-AKT1-Mediated Inward K+ Currents in Xenopus Oocytes.
Supplemental Figure 6. Interaction Analysis between Os-CIPKs and
Os-AKT1.
Supplemental Figure 7. Electrophysiological Analysis of Os-AKT1
Regulation by Other Os-CIPKs in HEK293 Cells.
Supplemental Figure 8. Electrophysiological Analysis of Os-AKT1
Activation by Cytosolic Ca2+ in HEK293 Cells.
Supplemental Figure 10. Phenotype of Rice Os-cbl1 Mutant.
Supplemental Figure 11. Os-AKT1-Mediated K+ Currents in HEK293
Cells under Different K+ Concentrations.
Supplemental Figure 12. The pH Sensitivity of AKT1 and Os-AKT1
Channels in Arabidopsis Root Cell Protoplasts.
Supplemental Figure 13. Interaction Analysis between At-KC1 and
Os-AKT1.
Supplemental Table 1. Primer Sequences Used in Real-Time PCR
Experiments.
Supplemental Table 2. Primer Sequences Used for Cloning.
Supplemental Data Set 1. Text File of the Alignment Used for the
Phylogenetic Analysis in Supplemental Figure 2.
ACKNOWLEDGMENTS
We thank Richard Gaber (Northwestern University) for providing yeast
strains R757 and R5421 that were used in the yeast complementation
experiments. We thank Emily Limam (University of Southern California)
for providing pGEMHE vector for the experiments using oocytes and Jörg
Kudla (Universität Münster, Germany) for providing the vectors pSPYNE(R)
173 and pSPYCE(MR) for BiFC assays and PM marker pGPTVII-CBL1nOFP. We thank Chuanqing Sun (China Agricultural University) for providing
the vector pUN1301 and for the assistance of rice planting in field. We also
thank Yongbiao Xue (Institute of Genetics and Developmental Biology,
Chinese Academy of Sciences) for providing the Os-CIPK23 RNAi lines.
This work was supported by grants from the “973” Project (2011CB100300
to Y.W.; 2012CB114203 to W.-H.W.), the National Natural Science
Foundation of China (No. 31270306 to Y.W.; No. 31121002 to W.-H.W.;
No. J1103520), and the “111” Project (No. B06003).
AUTHOR CONTRIBUTIONS
Y.W. and W.-H.W. designed the research. J.L., Y.L., G.-N.Q., J.L., and
Z.-J.X. performed research and analyzed data. J.L., Y.L., and Y.W. wrote
the article. Y.W. and W.-H.W. revised the article.
Received January 22, 2014; revised July 3, 2014; accepted July 21,
2014; published August 5, 2014.
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The Os-AKT1 Channel Is Critical for K+ Uptake in Rice Roots and Is Modulated by the Rice
CBL1-CIPK23 Complex
Juan Li, Yu Long, Guo-Ning Qi, Juan Li, Zi-Jian Xu, Wei-Hua Wu and Yi Wang
Plant Cell; originally published online August 5, 2014;
DOI 10.1105/tpc.114.123455
This information is current as of June 15, 2017
Supplemental Data
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