1. Introduction
As one of the most abundant nutrient ions, potassium (K
+) plays crucial roles in plant growth and development, including enzyme activation, osmotic regulation, electrical neutralization, and membrane potential maintenance [
1,
2,
3,
4]. However, the available K
+ concentration in soil is variable and relatively low for plant growth [
4,
5]. Therefore, plants often suffer low-K
+ stress in the natural environment. Under K
+-limited conditions, plants show leaf chlorosis phenotype, starting with older leaves, a typical K
+-deficient symptom; subsequently, plant growth and development are inhibited [
6,
7]. In agricultural production, K
+ deficiency will significantly reduce crop yield and quality [
3,
8]. Therefore, investigation of the mechanisms underlying how plants respond to low-K
+ stress will provide important insights into plant KUE (K utilization efficiency) improvement.
Plants absorb K
+ from the environment through a series of K
+ channels and transporters. In
Arabidopsis, the Shaker family inward K
+ channel AKT1 (
ARABIDOPSIS K
+ TRANSPORTER 1) and the KUP/HAK/KT family K
+ transporter HAK5 (HIGH-AFFINITY K
+ TRANSPORTER 5) are considered the most important components for root K
+ uptake [
9,
10,
11,
12,
13]. Their homologs in crops are also responsible for K
+ uptake, and they determine crop yield and stress resistance [
14,
15,
16]. NH
4+ is an important nitrogen source for plants. NH
4+ and K
+ display many similarities, such as hydrated diameters, charge, and influence on membrane potentials, which result in an antagonism between the two ions [
17,
18]. External NH
4+ inhibits HAK5-mediated high-affinity K
+ uptake, as well as represses the upregulation of the
HAK5 transcript under low K
+ conditions [
10,
19,
20]. By contrast, AKT1-mediated K
+ uptake is not affected by NH
4+ [
10].
Our previous study identified an AKT1-mediated K
+ uptake pathway in
Arabidopsis. The protein kinase LKS1/CIPK23 (LOW POTASSIUM SENSITIVITY 1/CBL-INTERACTING PROTEIN KINASE 23), interacting with the Ca
2+-binding proteins CBL1/CBL9, phosphorylates AKT1 to enhance K
+ uptake under low-K
+ stress [
11]. A similar regulatory pathway was also identified in rice [
14]. A recent study revealed that the CBL1/9-CIPK23 complex also phosphorylates HAK5 to promote high-affinity K
+ uptake under low-K
+ conditions [
21,
22]. Furthermore, the CBL1/9–CIPK23 complex can also regulate nitrate (NO
3−) and ammonium (NH
4+) uptake in
Arabidopsis root [
23,
24]. These studies demonstrate that LKS1/CIPK23 is essential for K
+/NO
3−/NH
4+ uptake and ion homeostasis under nutrient deficient conditions. However, how is LKS1/CIPK23 regulated? Low-K
+ stress can strongly induce
LKS1 transcripts in
Arabidopsis root, which is one of most important mechanisms in the plant response to K
+ deficiency [
11]. In addition, high NH
4+ upregulates
LKS1/CIPK23 transcripts, suggesting that the transcriptional regulation of
LKS1 is crucial for plants in response to both low K
+ and high NH
4+ to coordinate K
+/NH
4+ ion homeostasis. However, the transcriptional mechanism is little understood.
STOP1 (SENSITIVE TO PROTON RHIZOTOXICITY 1), a C2H2 zinc finger protein, functions as a crucial transcription factor that is involved in the
Arabidopsis response to low-pH and -aluminum (Al
3+) stresses by regulating the transcription of malate transporter
ALMT1 (ALUMINUM-ACTIVATED MALATE TRANSPORTER 1) and citrate transporter
MATE (MULTIDRUG AND TOXIC COMPOUND EXTRUSION) [
25,
26,
27]. Recent studies revealed that STOP1 can also participate in the regulation of root cell elongation under low Pi stress [
28,
29]. A growing number of studies have indicated that the STOP1-like proteins from rice (
Oryza sativa) [
30],
Eucalyptus [
31], buckwheat (
Fagopyrum esculentum Moench) [
32], rice bean (
Vigna umbellata) [
33], and tobacco (
Nicotiana tabacum) [
34] show similar functions in low-pH and -aluminum (Al
3+) tolerance. These homologs are much conserved and regulate similar downstream target genes in different plant species [
31,
32,
33,
34,
35]).
In the present study, using a high-throughput screening method, we identified the transcription factor STOP1 that positively regulates LKS1 transcription in Arabidopsis responses to both low-K+ and high-NH4+ stresses. The STOP1 transcript level is upregulated by low K+ but not high NH4+. STOP1 proteins accumulate in the nucleus under low-K+ or high-NH4+ conditions, and they directly bind to the LKS1 promoter to upregulate LKS1 transcription. Subsequently, LKS1 phosphorylates AKT1 and HAK5 to enhance K+ uptake, while it phosphorylates AMTs (HIGH-AFFINITY AMMONIUM TRANSPORTERS) to inhibit NH4+ uptake. The STOP1/LKS1 pathway plays crucial roles in regulating K+/NH4+ ion homeostasis to strengthen Arabidopsis tolerance to low-K+ stress.
3. Discussion
The protein kinase LKS1/CIPK23 has been reported to play essential roles in diverse signaling pathways. Under LK conditions, LKS1 phosphorylates the K
+ channel AKT1 [
11], as well as the K
+ transporter HAK5 [
21], to enhance K
+ uptake in
Arabidopsis root cells.
Therefore, the plant tolerance to LK stress is strengthened. In addition, LKS1/CIPK23 also regulates the activity of nitrate transporter NRT1.1/CHL1. NRT1.1 is involved in both high- and low-affinity NO
3− uptake [
37,
38]. Under low NO
3− concentrations, CIPK23 phosphorylates NRT1.1, via which NRT1.1 is converted into a high-affinity nitrate transporter to adapt the reduced NO
3− level [
23]. A recent report showed that CIPK23 also inhibits the activity of ammonium transporter AMT1;1/1;2 to avoid NH
4+ toxic accumulation under high-NH
4+ conditions [
24]. Therefore, LKS1/CIPK23 is a key component to coordinate potassium and nitrogen balance in plant roots. More importantly, the transcription of
LKS1/
CIPK23 can respond to external K
+, NO
3−, and NH
4+ concentrations [
11,
23,
24], suggesting that the transcriptional regulation of
LKS1/
CIPK23 is essential in plant response to external potassium and nitrogen levels. The present study not only reveals the transcriptional regulation in plant LK response, but also may provide some clues to understand nitrogen response.
Previous studies have demonstrated that STOP1 is essential for proton and Al
3+ tolerance by regulating the expression of malate transporter
ALMT1 (
ALUMINUM-ACTIVATED MALATE TRANSPORTER 1) and citrate transporter
MATE (
MULTIDRUG AND TOXIC COMPOUND EXTRUSION) [
25,
26,
39]. In addition, STOP1 also regulates root cell elongation under low Pi stress [
28]. In the present study, we demonstrate that STOP1 is involved in the low-K
+ response by regulating
LKS1/CIPK23 transcription. A recent report also found that STOP1 regulates salt and drought tolerance by modulating
LKS1/CIPK23 transcription [
40]. All this evidence supports a conclusion that STOP1 functions as a key node that controls ion homeostasis when plants are subjected to nutrient or ion stresses. The STOP1/LKS1 regulatory pathway may play more important roles in the plant nutrient/ion regulatory network.
STOP1 is an important component to regulate the transcription of downstream target genes; however, how is the
STOP1 gene or protein regulated? Under low pH or Al
3+ stresses, the transcriptional level of
STOP1 is not affected [
25]. In addition, the
STOP1 transcript is not changed under low-phosphorus conditions [
28,
29]. A recent study showed that the F-box protein RAE1 (Regulation of Atalmt1 Expression 1) directly interacts with STOP1 and controls the stability of STOP1 proteins through the 26S proteasome pathway in Al
3+ resistance [
41]. These studies suggest that STOP1 is regulated at the post-translational level rather than the transcriptional level. Here, we found that STOP1 protein is accumulated after low-K
+ or high-NH
4+ stresses, and the
RAE1 OE plants showed similar phenotypes to
stop1 mutant under our LK conditions (
Figure 8,
Figure 9 and
Figure S8E). It is noteworthy that the transcript level of
STOP1 was upregulated by low K
+ but not high NH
4+ (
Figure 8 and
Figure 9). Therefore,
STOP1 can be regulated at both transcriptional and post-translational levels. We suggest that the different regulatory mechanisms may depend on the different upstream stress signals. STOP1 is involved in Al
3+ resistance by upregulating
ALMT1 transcription. In the present study, we found that STOP1 participates in the LK response by elevating
LKS1 transcription. However, the
almt1 mutant did not show any sensitive or tolerant phenotype when grown on LK medium (
Figure S8C), suggesting that STOP1/ALMT1 and STOP1/LKS1 are two independent signaling pathways involved in different stress responses. In addition, 5 mM MES was added to LK medium to stabilize pH, and it could be seen that
stop1,
lks1 and
akt1 still showed LK sensitive phenotypes (
Figure S8F). The results showed that the sensitive phenotype of the
stop1 mutant was not mainly caused by the decreased pH in the medium.
A previous study showed that STOP1 regulates
ALMT1 transcription by binding to the sequence GGGGAGGGC in the
ALMT1 promoter [
42]. However, we found that the binding site of STOP1 on the
LKS1 promoter is the sequence CCTTCCTCG (
Figure 5). A recent study indicated that STOP1 can also bind to the
RAE1 promoter at the sequence CCTTCCTCG [
41], suggesting that STOP1 binds to the same
cis-element in the
LKS1 and
RAE1 promoters. In addition, this binding site was also confirmed by a recent report [
40]. Obviously, STOP1 regulates different target genes by binding to different
cis-elements in promoter regions. However, the mechanisms underlying how STOP1 responds to different stress signals and recognizes different
cis-elements still need to be further investigated.
According to the expression analyses, the transcripts of
LKS1 were extremely low in
stop1 mutants compared with wild type (
Figure 3A,
Figure 4D and
Figure 7A), suggesting that STOP1 should be the major transcription factor regulating
LKS1. In addition, the STOP1 homolog, STOP2, is not involved in the LK stress response, because the
stop2 mutant did not show any different phenotype compared with the wild type when grown on LK medium (
Figure S8D).
Low K
+ or high NH
4+ alone did not cause the obvious leaf chlorosis phenotype in
lks1 and
akt1 mutants in our transfer assays, although the K
+ content in mutant was significantly reduced (
Figure 8 and
Figure 9). The presence of NH
4+ could inhibit K
+ uptake, subsequently enhancing the leaf chlorosis phenotype of
akt1 and
lks1 mutants under low-K
+ conditions (
Figure S9A; [
13]). Therefore, NH
4+ used in the LK medium makes the phenotype more visible; however, it also causes NH
4+ toxicity. A recent study revealed that CIPK23 inhibits NH
4+ transport under NH
4+ toxic conditions [
24]. Here, our data also demonstrate that STOP1/LKS1 is involved in the NH
4+ response.
NH
4+ can inhibit primary root growth of plants under low-K
+ conditions [
9,
13]. Along with the increment in NH
4+, the primary root growth of wild-type and mutant plants (
stop1,
lks1, and
akt1) was gradually inhibited (
Figure S9A). Wild-type root growth was restricted in low-K
+ and high-NH
4+ conditions; however,
lks1 and
akt1 root could still grow (
Figure 2B) [
11]. Our previous study demonstrated that AKT1 is involved in low-K
+ sensing (presence of NH
4+) in
Arabidopsis root and subsequently regulates root growth by modulating PIN1 (PIN-FORMED 1) degradation and auxin redistribution in root [
43]. The
akt1 mutant root cannot respond to external LK stress;
lks1 and
cbl1 cbl9 mutants display a similar root phenotype in this regard. Therefore, loss of function of
AKT1,
LKS1, and
CBL1/9 leads to the root growth phenotype under this LK conditions. We noticed that the primary root of
stop1 mutants could not grow under LK conditions, which is similar to wild type. In
stop1 mutant,
LKS1 expression was significantly reduced (
Figure 3A), which impaired AKT1- and HAK5-mediated K
+ uptake (
Figure 6C, [
21]), resulting in a leaf chlorosis phenotype (
Figure 2A and
Figure S2F). However,
LKS1 expression did not completely disappear in the
stop1 mutant (
Figure 3A), and the low expression level could still activate partial AKT1 channels (
Figure 6A). Therefore,
stop1 mutant root could still respond to external LK and stop growth similar to wild type.
Both
LKS1 OE and
STOP1 OE showed an LK tolerant phenotype in shoot, which was due to increased K
+ content in their shoot (
Figure 3D,F). However, they displayed different root phenotypes. The
LKS1 OE accumulated more K
+ in root, which promoted root growth under LK conditions. On the other hand, the K
+ content in
STOP1 OE root was not significantly increased compared with wild type; therefore,
STOP1 OE showed a similar root phenotype to wild type (
Figure 3D,F). According to the root/shoot K
+ distribution in
STOP1 OE and
stop1/LKS1 OE lines (
Figure 3F and
Figure 4E), STOP1 may also positively regulate some other genes involved in root-to-shoot K
+ transport. In addition, both
lks1 mutant and
LKS1 OE plants showed root growth phenotypes under LK conditions (
Figure 2A and
Figure 3D); however, the mechanisms should be different. As discussed above, loss of function of
LKS1 leads to a root growth phenotype under this LK condition. The complementation line (
lks1-3/pLKS1:VENUS-LKS1) can restore this root phenotype to the wild-type level (
Figure S8B). Comparatively, overexpression of
LKS1 enhanced K
+ uptake and accumulation in root (
Figure 3F), which led to the root growth phenotype in the
LKS1 OE line. The
lks1/STOP1 OE and
stop1/LKS1 OE lines displayed
lks1 and
LKS1 OE root phenotypes, respectively (
Figure 4B,F). The
lks1 stop1 double mutant showed a root growth phenotype similar to the
lks1 mutant (
Figure S5). All this genetic evidence and these root phenotypes indicated that
LKS1 could be the downstream target of STOP1.
In the present study, we identified an essential transcription factor STOP1 that is involved in
Arabidopsis response to low-K
+ and high-NH
4+ stresses by regulating
LKS1 transcription. On the basis of previous studies, we extend the K
+/NH
4+ uptake regulatory network and propose a working model (
Figure 10). When plants are subjected to low-K
+ stress, the
STOP1 gene is somehow upregulated, and then STOP1 proteins directly bind to the
LKS1 promoter and induce
LKS1 transcription in the nucleus. Subsequently, LKS1 proteins are recruited to the PM by CBL1/CBL9 and phosphorylate the K
+ channel AKT1 and the K
+ transporter HAK5 to enhance K
+ uptake in plant roots. In addition, high-NH
4+ stress leads to STOP1 protein accumulation in nucleus, which positively regulates
LKS1 transcription. Then, the CBL1/LKS1 complex phosphorylates and represses the activities of NH
4+ transporters AMTs to inhibit excess NH
4+ uptake. Therefore, the STOP1/LKS1 pathway plays crucial roles in the K
+ and NH
4+ uptake/homeostasis, which coordinates potassium and nitrogen balance in plants responses to external fluctuating nutrient levels.
4. Materials and Methods
4.1. Plant Materials
The
Arabidopsis (
Arabidopsis thaliana) Columbia ecotype (Col-0) was used as the wild type in this study. The
Arabidopsis mutants
stop1-2 (T-DNA insertion line, SALK_114108) and
stop1-1 (derived from an ethyl methanesulfonate-mutagenized M2 population of Col-0 in a previous study) [
25] were obtained from the RIKEN Bio-Resource Center. The
lks1-3 (SALK_036154),
akt1 (SALK_071803), and
LKS1 OE lines were obtained as described previously [
11]. The
stop2 (SAIL_402_D03) and Col-3 lines were received from the Eurasian
Arabidopsis Stock Center (uNASC). The
almt1 (SALK_009629) line was described previously [
28].
RAE1 overexpressing plants (
RAE1 OE-7 and
RAE1 OE-21) were described previously [
41].
The
STOP1 coding sequence was cloned into the pCAMBIA1300 vector (Cambia) driven by the
STOP1 native promoter (2 kb), and then the vector was transformed into the
stop1-2 mutant to obtain the complementation lines (
COM-1 and
COM-2). The
pSuper:STOP1 vector was generated by cloning the
STOP1 CDS into pSuper1300 plasmid under the control of the
Super promoter [
44]. The
pSuper:STOP1 vector was transformed into Col-0 to acquire the
STOP1 OE (
OE-1 and
OE-2) transgenic plants. The
lks1-3/
STOP1 OE transgenic lines (
lks1-3/
STOP1 OE-1 and
lks1-3/
STOP1 OE-2) were constructed by transforming the
pSuper:STOP1 vector into the
lks1-3 mutant. In the processes of vector construction, Phusion
® HF DNA Polymerase (M0530L, BioLabs) was used to clone the gene fragments, and the TAKARA DNA ligation system was used to obtain the genetic constructs. The recombinant plasmid was introduced into
E. coli strain DH5α and the plasmid was extracted by AxyPrep
TM Plasmid Miniprep Kit (AXYGEN). The
stop1-1/
LKS1 OE line was generated by crossing
stop1-1 with
LKS1 OE. To construct the
lks1 stop1-2 double mutants using the Crispr/Cas9 genome editing technique, two targets (
Figure S5B) were designed within the
LKS1 genomic sequence on the website
http://www.genome.arizona.edu/crispr/CRISPRsearch.html (10 December 2019), and the two targets were used to design the primers (LKS1 DT1-BsF, LKS1 DT2-BsR, LKS1 DT1-F0, LKS1 DT2-R0, as mentioned in
Supplementary Table S1) for Crispr/Cas9 [
45]. The
lks1 stop1-2 double mutants were produced by transforming the Crispr/Cas9 construct into
stop1-2 mutant. Genomic DNA from plants in the T
1 generation was sequenced for construct verification.
Arabidopsis transformation with
Agrobacterium (strain GV3101) was carried out by the floral dip method [
46].
Accession Numbers: Sequence data for the genes described in this article can be found in the
Arabidopsis TAIR database (
https://www.arabidopsis.org (3 January 2021)) under the following accession numbers: AT1G34370 for
STOP1, AT1G30270 for
LKS1/
CIPK23, AT5G22890 for
STOP2, AT2G26650 for
AKT1, AT1G08430 for
ALMT1, and AT1G80670 for
RAE1.
4.2. Phenotypic Analyses and Growth Conditions
The Arabidopsis seeds were surface-sterilized using 6% (v/v) NaClO and incubated at 4 °C in darkness for 3 days. Then, the seeds were germinated on MS (Murashige & Skoog) medium at 22 °C under constant illumination at 60 μmol·m–2·s–1. All the medium used in this study contained 0.9% (w/v) agar (Ourchem) and 3% (w/v) sucrose (Sinopharm).
For the LK phenotype test, 5 day old seedlings grown on MS medium (20 mM K
+) were transferred to LK medium (0.1 mM K
+) or MS medium for 10 days (to observe the LK sensitive phenotype) or 12 days (to observe the LK tolerant phenotype). The LK medium was made by modifying the MS medium described previously [
11]. The MS medium contained 1.5 mM MgSO
4, 2.99 mM CaCl
2, 20.6 mM NH
4NO
3, 18.79 mM KNO
3, and 1.25 mM KH
2PO
4, while the LK medium contained 1.5 mM MgSO
4, 2.99 mM CaCl
2, 28.75 mM NH
4NO
3, and 1.25 mM NH
4H
2PO
4. The final K
+ concentration in the LK medium was adjusted to 0.1 mM by adding KCl. In the present study, LK referred to the low-K
+ medium as described above, unless the concentrations were indicated.
For phenotypic assays on high-K
+ (20 mM) and low-K
+ (0.1 mM) medium in the absence of NH
4+ (
Figure 8), 5 day old seedlings grown on high-K
+ (20 mM) medium were transferred to high-K
+ (20 mM) and low-K
+ (0.1 mM) medium for 10 days. The low-K
+ (0.1 mM) medium contained 1.5 mM MgSO
4, 1.25 mM H
3PO
4, 2.99 mM Ca(NO
3)
2, and 0.1 mM KCl. The high-K
+ (20 mM) medium was supplemented with KCl to 20 mM.
For phenotypic assays on high-NH
4+ (30 mM) and low-NH
4+ (0 mM) medium (
Figure 9), 5 day old seedlings grown on low-NH
4+ (0 mM) medium were transferred to high-NH
4+ (30 mM) and low-NH
4+ (0 mM) medium for 10 days. The low-NH
4+ (0 mM) medium contained 1.5 mM MgSO
4, 1.25 mM H
3PO
4, 2.99 mM Ca(NO
3)
2, and 20 mM KCl. The high-NH
4+ (30 mM) medium was supplemented with 30 mM NH
4Cl. Other microelements in the above medium were consistent with MS medium.
For the phenotypic test, each plate contained four seedlings for each plant material. In one independent experiment, there were at least three biological replicates (three plates), and each phenotype test was performed at least three times.
For seed harvesting, Arabidopsis plants were cultured in the potting soil mixture (rich soil/vermiculite = 2:1, v/v) and kept in growth chambers (temperature was 22 °C, illumination was 120 μmol·m−2·s−1, and the relative humidity was approximately 70%) with long-day conditions (16 h light/8 h darkness).
4.3. K+ Content Measurement
The 5 day old Arabidopsis seedlings grown on MS medium were transferred to LK or MS medium and treated for the indicated times described in figure legends. For low-K+ treatment, 80 to 100 individual seedlings from two plates were collected as one biological replicate. For the K+ sufficient treatment, 40 to 50 individual seedlings from one plate were collected as one biological replicate. The shoots and roots were harvested separately. Three or four biological replicates (n = 3 or n = 4) were used in one independent experiment. To test the plants with the LK sensitive phenotype, K+ content was measured after 7 days of LK treatment. To test the plants with LK tolerant phenotype, K+ content was measured after 10 days of LK treatment.
To test the K
+ content under low-K
+ (0 mM NH
4+) (
Figure 8B) or high-NH
4+ (
Figure 9B) conditions, the 5 day old seedlings were transferred to low-K
+ or high-NH
4+ medium for 7 days. A total of 40 to 80 individual seedlings were collected as one biological replicate, and the shoots and roots were harvested separately. Three or four biological replicates (
n = 3 or
n = 4) were used in one independent experiment.
The collected samples were dried at 80 °C for 24 h to a constant weight, and then the dry weight was measured. The samples were treated in a muffle furnace at 300 °C for 1 h and then 575 °C for 5 h. The ashes were dissolved and diluted in 0.1 N HCl. The K+ concentrations were measured using the 4100-MP AES device (Agilent).
4.4. Transcription Analyses
For both RT-PCR and RT-qPCR analyses, total RNA was extracted from the roots of 7 day old seedlings using Trizol reagent (Invitrogen). The roots of 200 individual seedlings from two plates were collected and used as one biological replicate. Three or four biological replicates (n = 3 or n = 4) were used in one independent experiment. Then, 8 μg of total RNA was treated with DNase I (RNase Free, Takara) at 37 °C for 30 min and then 65 °C for 10 min to eliminate DNA contamination. Next, 4 μg of DNase I-treated RNA was used to synthesize complementary DNA (cDNA) with SuperScriptII RNase reverse transcriptase (Invitrogen). Oligo (dT) primers (Promega) were used for RT-PCR and Random Hexamer primers (Promega) were used for quantitative real-time PCR analyses (RT-qPCR).
For RT-PCR in
Figure S2D, the
EF1α gene was used as an internal standard for normalization of gene expression levels. The primers used to detect the CDS of
STOP1 are shown in
Supplementary Table S1. PCR was performed for 30 cycles, each with 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min 50 s.
For RT-qPCR, the cDNA was diluted 40-fold with double-distilled water, and 8 μL of diluted cDNA was used as the template in each reaction. The Power SYBR Green PCR Master Mix (Applied Biosystems, USA) was used to carry out this assay. Herem 20 μL was one reaction volume containing 10 μL of SYBR Green premix, 8 μL of cDNA, and 2 μL of forward and reverse primers (1 μM), which was reacted on a 7500 Real Time PCR System machine (Applied Biosystems). The PCR was conducted as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. To normalize the test gene expression levels,
ACTIN2/8 was used as an internal standard. The primers used in this experiment are listed in
Supplementary Table S1.
For the LK induction experiment in
Figure 7A, 5 day old seedlings were transferred to MS or LK medium for 1 day. For RT-qPCR analyses of
STOP1 and
LKS1 expression in low-K
+ response in the absence of NH
4+ (
Figure 8C), 5 day old seedlings were transferred to high-K
+ (20 mM) and low-K
+ (0.1 mM) medium for the indicated times. For RT-qPCR analyses of
STOP1 and
LKS1 expression in the high NH
4+ response (
Figure 9C), 5 day old seedlings were transferred to high-NH
4+ (30 mM) and low-NH
4+ (0 mM) medium for the indicated times. The roots of 200–240 individual seedlings were collected and used as one biological replicate for RT-qPCR assays. Three or four biological replicates (
n = 3 or
n = 4) were used in one independent experiment.
4.5. Mating-Based Y1H Screening
The LKS1 promoter region was divided into four fragments, F1 (−382 to −1 bp), F2 (−757 to −364 bp), F3 (−1078 to −738 bp), and F4 (−1506 to −1061 bp), and the four fragments were constructed into the pHISi-1 vector, used as baits. For the mating-based Y1H screening, all the GAL4-AD-TF strains were grown overnight in SD/-Trp medium in 2 mL 96-well plates. Yeast strains YM4271 carrying the baits were grown overnight at the same time. Then, 20 μL of donor and host strains were transferred to a new 2 mL 96-well plate with 100 μL of YPAD medium. Mating was carried out for 24 h by shaking at 30 °C. After dilution with 1.5 mL of water, the mating products were plated on SD/-Trp-His selective plates and incubated for 3 days at 30 °C. Then, 5 mM or 10 mM 3-amino-1, 2, 4-triazole (3-AT) was added to SD/-Trp-His selective plates (Sigma-Aldrich).
4.6. Vector Constructions and Yeast One-Hybrid Assays
The
pLKS1:LacZ construct was generated by cloning the
LKS1 promoter fragment (1506 bp) into pLacZi2μ vector [
47]. To generate various
LacZ reporter genes driven by the sub-fragments of the
LKS1 promoter shown in
Figure 1B, the promoter fragments were amplified by PCR using
pLKS1:LacZ construct as the template. The respective pairs of primers are shown in
Supplementary Table S1. In order to generate the four types of mutants in the F2-2 fragment shown in
Figure 5G, the QuikChange Site-Directed Mutagenesis Kit (Agilent) was used to conduct point mutation PCR, and the F2-2 fragment fused with
LacZ reporter gene vector was used as the template. The reaction volume and PCR program referred to the manufacturer’s instructions. To generate
AD-STOP1, the
STOP1 CDS was amplified by PCR and then cloned into the pB42AD vector (Clontech). To construct
AD-STOP1H266Y vector, the template
AD-STOP1 plasmid and the QuikChange Site-Directed Mutagenesis Kit (Agilent) were used to produce the single-base mutation. The
LKS1 promoter fragments were co-transformed separately with
AD-STOP1 into the yeast strain EGY48. The transformed strains were cultured on SD/-Trp-Ura plates and confirmed by PCR. Then, these transformants were grown on proper SD/-Trp-Ura plates containing X-gal (5-bromo-4-chloro-3-indolyl-β-
d-galactopyranoside), 2% galactose, and 1% raffinose for blue color development. The yeast transformation assay was conducted as described in the Yeast Protocols Handbook (Clontech).
4.7. Microscopy Imaging
The
stop1-2/
pSTOP1:GFP-STOP1 and
stop1-2/
pUBQ:GFP-STOP1 [
28] plants were used to observe the fluorescence of GFP-STOP1. The
lks1-3/
pLKS1:VENUS-LKS1 plants was constructed by transforming the
pLKS1:VENUS-LKS1 vector into
lks1-3 mutant. To observe the fluorescence induced by LK stress, the 5 day old seedlings of
stop1-2/
pSTOP1:GFP-STOP1,
stop1-2/
pUBQ:GFP-STOP1, and
lks1-3/
pLKS1:VENUS-LKS1 plants were transferred to MS or LK medium for 1 day. To observe the fluorescence under low-K
+ or high-NH
4+ treatment (
Figure 8 and
Figure 9), the 5 day old seedlings were transferred to low-K
+ or high-NH
4+ medium for the indicated times. Then, the seedlings were used for the fluorescence observation. To show the outline of the root cells, seedlings were dipped in 30 μM propidium iodine (Sigma-Aldrich) solution for 1 min at room temperature and rinsed twice with double-distilled water. Images were collected on a Zeiss LSM710 confocal microscope using a Plan Apochromat ×40/1.4 Oil DIC M27 objective. GFP-STOP1 and PI were excited sequentially with a blue argon ion laser (488 nm, 45% strength) and a DPSS laser (561 nm, 1% strength). Emitted light was collected from 493 to 556 nm for GFP-STOP1 and from 647 to 721 nm for PI. Venus-LKS1 was excited with the blue argon ion laser (488 nm, 60% strength), and the emitted light was collected from 493 to 556 nm. The fluorescence intensity was measured by the Image J program. The photos of the experimental group and control group were taken with the same microscope and camera settings.
4.8. GUS/LUC Assay
pLKS1:GUS was constructed by cloning the LKS1 promoter fragment (1.5 kb) with a β-glucuronidase (GUS) coding sequence into pCAMBIA1381 (Cambia) vector. The pSuper:STOP1 and pLKS1:GUS vector were transfected into Agrobacterium (strain GV3101). Agrobacterium cells were harvested by centrifugation and suspended in the solutions containing 10 mM MES pH 5.6, 10 mM MgCl2, and 200 μM acetosyringone to an optical density (OD600) of 0.8, incubated at 28 °C for 2 h, and then the bacterial solution was injected into the Nicotiana benthamiana leaves. The bacterial solution was mixed in the following proportions: 300 μL of P19, 400 μL of pLKS1:GUS, 500 μL of pSuper:STOP1, and 4 μL of pSuper:LUC (luciferase). In the control group, pSuper:STOP1 was replaced by Super1300. The GUS and LUC activity was measured after 3 days of injection. The GUS activity was measured using methyl umbelliferyl glucuronide (Sigma-Aldrich) and an F-4500 Flourescence Spectrophotometer (Hitachi). LUC activity was used as an internal control and measured by GloMax® 20/20 Luminometer (Promega). The GUS/LUC ratio was used to determine the STOP1 binding activity to the LKS1 promoter.
4.9. GUS Staining Assay
The pLKS1:GUS or pSTOP1:GUS vectors were generated by fusing the LKS1 promoter fragment (1.5 kb) or STOP1 promoter fragment (2 kb) with the β-glucuronidase (GUS) coding sequence into pCAMBIA1381 (Cambia). Then, the pLKS1:GUS vector was transformed into Col-0 and stop1-1 to obtain the pLKS1:GUS and stop1-1/pLKS1:GUS transgenic plants. pSTOP1:GUS was obtained by transforming the pSTOP1:GUS vector into Col-0. For GUS staining assays, pSTOP1:GUS was incubated in GUS staining buffer for 15 min, and pLKS1:GUS or stop1-1/pLKS1:GUS was stained for 50 min at 37 °C. Here, 10 mL of GUS staining buffer contained 5 mg of X-gluc (BIOSYNTH, B-7300), 10–20 μL of N,N-dimethylformamide, 7.98 mL of PBS buffer (0.1 M, KH2PO4 and K2HPO4 with pH of 7.0), 1 mL of 5 mM potassium hexacyanoferrate (III), 1 mL of 5 mM potassium hexacyanoferrate (II), and 10 μL of Triton X-100.
4.10. ChIP-qPCR Assay
To construct
pSuper:STOP1-MYC, the
STOP1 coding sequence fused with the MYC label was driven by the
Super promoter and then transformed into Col-0. The
pSuper:STOP1-MYC transgenic plant used in the ChIP assay was a single-copy homozygous line. The roots of 12 day old
pSuper:STOP1-MYC seedlings (about 0.5–1 g fresh weight) were harvested and crosslinked with 1% formaldehyde for the ChIP experiment. Then, the nuclei were isolated, and the extracted nuclei were lysed according to [
48] with minor modifications in lysis buffer composition (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.1% SDS, 0.1% Na deoxycholate, 1% Triton X-100, 1 μg·mL
−1 pepstain A, 1 μg·mL
−1 aprotinin). After the DNA was sheared, the sample was incubated with anti-MYC antibody (Abmart) to immunoprecipitate protein/DNA complexes. After reverse crosslinking and protein digestion, the precipitated DNA was used for qPCR detection. The primers are listed in
Supplementary Table S1. qPCR was performed for 40 cycles, each at 95 °C for 15 s, 55 °C for 20 s, and 60 °C for 45 s. The detailed experimental methods were described previously [
48].
4.11. EMSA (Electrophoretic Mobility Shift Assays)
The STOP1 and STOP1C796T coding sequence was cloned into the pET-30a (+) vector (Novagen) to obtain the STOP1 and STOP1H266Y protein expression plasmid. The recombinant plasmid was introduced into E. coli strain BL21. E. coli cells were induced with 0.2 mM IPTG overnight at 18 °C and collected by centrifugation (3220× g) at 4 °C for 10 min (Eppendorf centrifuge 5810 R with A-4-62 rotor). After the bacterial precipitation was cleaned once with His-Binding Buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 30 mM Imidazole), the precipitation was suspended with 10 mL of His-Binding Buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 30 mM Imidazole, 1 mM PMSF, 1 mM DTT), and the ultrasonication was performed on the bacterium suspension. A total of 99 ultrasounds were performed using 200 W of power for a 2 s ultrasound time and a 4 s interval. The ultrasonic solution was centrifuged (18,514× g) at 4 °C for 30 min (Eppendorf centrifuge 5810 R with F-34-6-38 rotor), and the supernatant of the bacteria lysate was used for protein purification. The protein was purified using Ni-Sepharose 6 Fast Flow (GE Healthcare). The wash buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 50 mM Imidazole) was used to wash Ni-Sepharose, and eluting buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 250 mM Imidazole) was used to elute protein. The protein concentration was determined by Bio-Rad protein assay and 250 μg of purified protein was used in EMSA experiment. The EMSA was conducted using LightShift Chemiluminescent EMSA Kit (ThermoFisher) according to the manufacturer’s protocol. The probes of the LKS1 promoter were obtained by PCR using biotin-labeled or biotin-unlabeled primers. Biotin-unlabeled probes of the same sequences were used as competitors and His protein was used as the negative control.
4.12. Patch-Clamp Whole-Cell Recording from Root-Cell Protoplasts
Root-cell protoplasts were isolated by enzyme solution from 5 day old primary roots of
Arabidopsis seedlings. The enzyme solution containing 1.5% (
w/
v) cellulysin (Calbiochem), 1.5% (
w/
v) cellulase RS (Yakult Honsha Co.), 0.1% (
w/
v) pectolyase Y-23 (Seishin Pharmaceutical Co.), and 0.1% (
w/
v) BSA was dissolved in standard solution containing 10 mM K
+ glutamate, 2 mM MgCl
2, 1 mM CaCl
2, 350 mM sorbitol, and 5 mM MES (pH 5.8 adjusted with Tris). The primary roots were cut into small pieces and incubated in the enzyme solution at 23 °C for 40 min to release root-cell protoplasts. The protoplasts were filtered through 80 μm nylon mesh and washed twice with standard solution by centrifugation at 160×
g for 5 min. The isolated root-cell protoplasts were kept on ice before patch-clamp experiments. The patch-clamp experiments were recorded using an Axopatch 200B amplifier (Axon Instruments) at room temperature in dim light. The contents of the bath and pipette solutions were the same as described previously [
11].
4.13. Kinetic Analysis of K+ Uptake
Arabidopsis seeds were germinated on MS medium at 22 °C under constant illumination. For K
+ depletion experiments, 6 day old seedlings were collected (0.6 g of fresh weight used as one biological replicate) and pretreated in one-quarter-strength MS solution (5.15 mM NH
4NO
3, 0.375 mM MgSO
4, 4.7 mM KNO
3, 0.31 mM KH
2PO
4, 0.75 mM CaCl
2, and 5 mM MES, pH 5.8 adjusted with Tris) at 22 °C overnight. Then, the seedlings were transferred into 25 mL of K starvation solution (200 μM CaSO
4, 5 mM MES, pH 5.8 adjusted with Tris) for 2 days. During these 2 days, K starvation solution was changed three times a day. Next, the seedlings were transferred to 25 mL of K depletion solution (200 μM CaSO
4, 250 μM KNO
3, 5 mM MES, pH 5.8 adjusted with Tris). All samples were shaken on a shaking table at 22 °C under constant illumination during the experiments [
11]. The solution samples were collected at different timepoints indicated in
Figure 6. The K
+ concentrations were measured using the 4100-MP AES device (Agilent).
4.14. Statistical Analyses
Data were shown as means ± SE. Student’s t-test was used to analyze statistical significance between treatment and control. The p-value was shown as * p < 0.05 or ** p < 0.01 to indicate significant differences.