Receptor-Like Protein Kinase Bak1 Promotes K Uptake by Regulating H - Atpase Aha2 Under Low Potassium Stress

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1 Receptor-like protein kinase BAK1 promotes K+ uptake by regulating
2 H+-ATPase AHA2 under low potassium stress

3
4 Zhi-Fang Wang1, Zhong-Mei Xie1, Ya-Lan Tan1, Jia-Ying Li1, Feng-Liu Wang1, Dan Pei1, Zhen
5 Li1, Yan Guo1, Zhi-Zhong Gong1,2, Yi Wang1*
6
7 1 State Key Laboratory of Plant Physiology and Biochemistry (SKLPPB), College of Biological
8 Sciences, China Agricultural University, Beijing 100193, China.
9 2 School of Life Science, Institute of Life Science and Green Development, Hebei University,
10 Baoding, Hebei, 071002, China.
11 ORCID ID: 0000-0002-3660-5859 (Y.W.)
12
13 Running title:
14 BAK1 phosphorylates AHA2 and promotes K+ uptake
15
16 The author responsible for distribution of materials integral to the findings presented in this
17 article in accordance with the policy described in the Instructions for Authors
18 (https://academic.oup.com/plphys/pages/General-Instructions) is Yi Wang (yiwang@cau.edu.cn).
19
20 Author Contributions
21 Y.W. designed the research. Z.F.W., Z.M.X., Y.L.T., J.Y.L. and F.L.W. conducted the experiments.
22 D.P., Z.L., Z.G. and Y.G. provide experimental support. Z.F.W. and Y.W. wrote and revised the
23 article.
24
*
25 Address correspondence to yiwang@cau.edu.cn.
26
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27 One-Sentence Summary: The receptor-like protein kinase BAK1 phosphorylates the
28 H+-ATPase AHA2 and enhances its activity, which subsequently promotes root K+ uptake under
29 low K+ conditions in Arabidopsis.
30
31 ABSTRACT
32 Potassium (K+) is one of the essential macronutrients for plant growth and development.
33 However, the available K+ concentration in soil is relatively low. Plant roots can perceive low K+
34 (LK) stress, then enhance high-affinity K+ uptake by activating H+-ATPases in root cells, but the
35 mechanisms are still unclear. Here, we identified the receptor-like protein kinase BAK1
36 (Brassinosteroid Insensitive 1-Associated Receptor Kinase 1) that is involved in LK response by
37 regulating the Arabidopsis (Arabidopsis thaliana) PM (plasma membrane) H+-ATPase isoform 2
38 (AHA2). The bak1 mutant showed leaf chlorosis phenotype and reduced K+ content under LK
39 conditions, which was due to the decline of K+ uptake capacity. BAK1 could directly interact
40 with the AHA2 C terminus and phosphorylate T858 and T881, by which the H+ pump activity of
41 AHA2 was enhanced. The bak1 aha2 double mutant also displayed a leaf chlorosis phenotype
42 that was similar to their single mutants. The constitutively activated form AHA2Δ98 and
43 phosphorylation-mimic form AHA2T858D or AHA2T881D could complement the LK sensitive
44 phenotypes of both aha2 and bak1 mutants. Together, our data demonstrate that BAK1
45 phosphorylates AHA2 and enhances its activity, which subsequently promotes K+ uptake under
46 LK conditions.
47
48 Keywords: Low potassium, receptor-like protein kinase, BAK1, AHA2, Arabidopsis

49
2
50 INTRODUCTION
51 As one of macronutrients, potassium (K) is essential for plant growth and development. Plants
52 need to absorb a large amount of K+ from external environment to maintain normal life activities.
53 Potassium constitutes 2%-10% of plant dry weight. The cytoplasmic K+ concentration in living
54 plant cells can reach 100-200 mM (Clarkson and Hanson 1980; Leigh and Wyn Jones, 1984;
55 Maathuis, 2009; Sodek., et al, 1980). However, in soils the available K+ for plants is relatively
56 low and the concentration is typically within micromolar range (Schroeder et al., 1994; Maathuis,
57 2009). Therefore, plants often suffer low K+ stress in natural environment. It is known that plants
58 can sense and transduce the low-K+ (LK) stress signal, and actively respond to LK stress for their
59 survival via physiological and morphological alteration (Xu et al., 2006; Ragel et al., 2015;
60 Wang and Wu, 2013; Wang and Wu, 2015; Wang and Wu, 2017).
61 As we know, enhancement of high-affinity K+ uptake is one of the most important
62 mechanisms in plant responses to LK stress (Wang and Wu, 2013). In Arabidopsis (Arabidopsis
63 thaliana), the K+ channel AKT1 (Arabidopsis K+ Transporter 1) and K+ transporter HAK5
64 (High-Affinity K+ Transporter 5) are considered as the two most important components for root
65 K+ uptake, and they mediate 80% K+ uptake under LK conditions. (Hirsch et al., 1998; Spalding
66 et al., 1999; Xu et al., 2006; Gierth et al., 2005; Pyo et al., 2010). Both AKT1 and HAK5 are
67 regulated by CBL1/9 (Calcineurin B-Like Protein 1/9)-CIPK23 (CBL-Interacting Protein Kinase
68 23) via phosphorylation to promote K+ uptake (Xu et al., 2006; Ragel et al., 2015; Scherzer et al.,
69 2015). The K+ uptake mediated by AKT1 channel is dependent on K+ gradients and
70 hyperpolarized membrane potential that is mainly established by PM (plasma membrane)
71 H+-ATPase. While, as a H+/K+ symporter, HAK5-mediated K+ uptake also depends on the H+
72 gradient across root cell plasma membrane. Therefore, proton concentration gradient and electric
73 potential gradient, also called protonmotive force (PMF), is the main driving force that energizes
74 the K+ uptake from soils against the K+ concentration gradient (Haruta and Sussman, 2012;
75 Sussman, 1994). The PMF is established and maintained primarily by H+-ATPase proteins
76 located at the PM (Haruta and Sussman, 2012; Sussman, 1994). Under LK conditions, the
3
77 activity of PM H+-ATPase is increased to enhance PMF that promotes K+ uptake
78 (Nieves-Cordones et al., 2014).
79 In Arabidopsis, there are 11 functional PM H+-ATPase genes, denoted AHA1 to AHA11
80 (Baxter et al., 2003). However, only AHA1 and AHA2 play dominant roles in Arabidopsis growth
81 and development. They show the highest expression levels among all the AHA genes (Haruta et
82 al., 2010). Double mutants of these two genes showed embryonic death (Haruta et al., 2010).
83 Compared with aha1 mutant, aha2 mutant mainly showed decreased PMF in root (Haruta et al.,
84 2010), suggesting that AHA2 is mainly involved in the formation of PMF in root and promotes
85 solute uptake. When external K+ concentration is decreased, the root cell membrane potential is
86 hyperpolarized within a few minutes (Maathuis and Sanders, 1993). It is speculated that AHA2 is
87 somehow activated under LK conditions, which is an essential step to enhance AKT1- and
88 HAK5-mediated high-affinity K+ uptake. However, the molecular mechanisms are still unclear.
89 More and more studies have shown that receptor-like protein kinases (RLKs) play important
90 roles in plant responses to environmental stimuli and developmental signals (Liu et al., 2020; Ma
91 et al., 2016; Liang and Zhou, 2018). PM-located BAK1 (Brassinosteroid Insensitive
92 1-Associated Receptor Kinase 1) is one of the most extensively studied RLK members in
93 Arabidopsis (Kim et al., 2013; Chinchilla et al., 2007; Heese et al., 2007; Postel et al., 2010;
94 Schulze et al., 2010; Sun et al., 2013; Yamada et al., 2016). BAK1, also known as SERK3,
95 belongs to the SERK family (Belkhadir et al., 2014). In many signal perception and transmission
96 processes, BAK1 acts as a co-receptor and directly interacts with different LRR-RLK receptors
97 (e.g. BRI1, FLS2, PSKR, ERECTAs, and so on) involved in various signal transduction
98 processes (Li et al., 2002; Nam and Li, 2002; Chinchilla et al., 2007; Heese et al., 2007; Ladwig
99 et al., 2015; Meng et al., 2015).
100 The present study demonstrates that BAK1 is also involved in the regulation of K+ uptake in
101 Arabidopsis response to LK stress. Under LK conditions, BAK1 directly interacts with and
102 phosphorylates AHA2 to enhance its activity. As a result, the transmembrane PMF is
103 strengthened, which promotes AKT1- and HAK5-mediated K+ uptake in root cells. The
4
104 BAK1-AHA2-AKT1/HAK5 pathway plays crucial roles in K+ absorption to strengthen
105 Arabidopsis tolerance to low K+ stress.
106 RESULTS
107 The bak1 mutants are sensitive to LK stress
108 Receptor-like protein kinases (RLKs) located at the plasma membrane (PM) play crucial roles in
109 the signal sensing and transduction of various physiological processes, including plant growth,
110 development, immunity, and reproduction (Liang and Zhou, 2018). However, whether and how
111 RLKs contribute to the sensing and adaptation to low-K+ (LK) stress are still elusive. In order to
112 investigate their functions in LK signaling, we screened a collection of about 200 T-DNA
113 insertion mutants of Arabidopsis RLKs by observing their phenotypes under LK conditions. We
114 determined that the mutant plants (bak1-3 and bak1-4) of the BAK1 gene displayed a leaf
115 chlorosis phenotype when grown on LK (80 μM K+) medium for 12 d (Figure 1A and S1A). In
116 addition, the root growth of mutant plants was also impaired (Figure 1A). Under high-K+ (HK, 5
117 mM) conditions, bak1-3 and bak1-4 mutants did not show different phenotypes compared with
118 wild-type plants (Col). The results of K+ content measurement showed that the shoot K+ contents
119 of bak1-3 and bak1-4 were lower than that of wild type under LK conditions (Figure 1B), which
120 was consistent with the shoot sensitive phenotype of these mutants on LK medium.
121 To further investigate the relationship between LK stress and leaf chlorosis phenotype, we
122 examined the phenotypes of bak1 mutants at different external K+ concentrations. The results
123 indicated that the sensitive phenotype of bak1 mutants was gradually restored along with the
124 increment of external K+ concentration in the medium (Figure S1D).
125 We also constructed the complementation lines of bak1-3 mutant (bak1-3/pBAK1:BAK1)
126 using BAK1 genomic sequence driven by BAK1 native promoter. BAK1 expression levels in the
127 two independent transgenic lines (COM1 and COM2) were reverted to wild-type level (Figure
128 S1B). Phenotypic test showed that COM1 and COM2 could fully complement the sensitive
129 phenotype of bak1-3 mutant on LK medium (Figure 1C). In addition, the K+ contents of the two
130 complementation lines were also restored (Figure 1D). These data indicate that loss-of-function
5
131 of BAK1 leads to the reduction of plant K+ content, which results in the LK sensitive phenotype.
132
133 BAK1 is involved in the K+ uptake regulation

134 We noticed that the total K+ amounts in bak1 mutants were significantly reduced compared with
135 wild type under LK conditions (Figure 2A), suggesting that BAK1 may be involved in the
136 regulation of K+ uptake, especially under LK conditions. Then, K+ depletion experiments were
137 carried out to verify the K+ uptake capacity of bak1 mutants (Figure S2A). As shown in Figure
138 2B, the root K+ uptake rates of bak1 mutants were substantially reduced compared to wild type.
139 Here, the K+ uptake defective mutant akt1 was used as control, whose K+ uptake rate was lower
140 than bak1 mutants (Figure 2B). These results indicate that BAK1 could positively regulate root
141 K+ uptake.
142 As we know, AKT1 and HAK5 are mainly responsible for K+ uptake in Arabidopsis roots
143 under LK conditions (Pyo et al., 2010). We wondered whether BAK1 directly regulated the
144 activities of AKT1 and HAK5 to participate in K+ absorption. Then, we first compared the
145 phenotypes of akt1, hak5, and bak1 mutants under LK conditions. The akt1 mutant showed more
146 severe sensitive phenotype than bak1 mutant. In addition, the akt1 bak1 double mutant exhibited
147 more sensitive phenotype than their single mutants (Figure 2C). These phenotype observations
148 indicate that BAK1 may regulate AKT1 activity. However, BAK1 and AKT1 did not show direct
149 protein-protein interaction in Nicotiana benthamiana leaves (Figure S3A and S3H). The results
150 of TEVC (two electrode voltage clamp) in Xenopus oocytes indicated that BAK1 did not affect
151 the channel activity of AKT1 in the presence or absence of CIPK23 and CBL1 (Figure S3B and
152 S3C).
153 As shown in Figure 2D, hak5 mutant showed weak sensitive phenotype on LK medium
154 compared with bak1 mutant. Considering that HAK5 mediates high-affinity K+ uptake at lower
155 K+ concentrations, we also tested the phenotypes of bak1 and hak5 mutants on the medium
156 containing 10 or 20 μM K+ (Pyo et al., 2010). Both bak1 and hak5 mutants displayed similar
157 short root phenotypes under these conditions (Figure S3D and S3E). However, BAK1 did not
6
158 directly interact with HAK5 either (Figure S3F and S3I). These data suggest that BAK1 may
159 regulate both AKT1- and HAK5-mediated K+ uptake in an indirect manner.
160
161 BAK1 may regulate AHA2 activity

162 It is well known that AKT1- and HAK5-mediated K+ uptake are dependent on the
163 transmembrane potential and H+ gradient that are mainly established by the PM H+-ATPases
164 (Haruta and Sussman, 2012; Nieves-Cordones et al., 2014; Palmgren, 2001). Therefore, it was
165 speculated that BAK1 might indirectly affect K+ uptake by regulating H+-ATPase activity at the
166 PM. Then, the phenotypes of aha1 and aha2 mutants on the LK medium were observed, since
167 AHA1 and AHA2 are two major functional members of the PM H+-ATPase family (Haruta et al.,
168 2010). As shown in Figure 3A and Figure S4A, only aha2 but not aha1 mutants displayed an
169 obvious leaf chlorosis phenotype under LK conditions. In order to test whether BAK1 affected
170 AKT1- and HAK5-mediated K+ uptake by regulating AHA2, the phenotypes of these mutant
171 plants were observed on LK medium. As shown in Figure 3B, akt1 hak5 double mutant could not
172 germinate on this LK medium, suggesting AKT1 and HAK5 are major components involved in
173 K+ uptake. Among these mutant plants, only aha2 mutant displayed similar phenotype like bak1
174 mutant under LK (10, 20, 80 μM) conditions (Figure 3B and S3D). To verify whether BAK1 and
175 AHA2 functioned in same signaling pathway, the aha2-6 bak1-5 double mutant was constructed
176 by crossing. Phenotype test indicated that the double mutant exhibited leaf chlorosis phenotype
177 and reduced shoot K+ content similar to their single mutants under LK conditions (Figure 3C and
178 3D), suggesting that they may work together to regulate K+ uptake.
179 Since AHA2 conducts H+ efflux across the PM, the H+ flux rates at root cell surface were
180 determined by using NMT assay. Indeed, the H+ efflux rate in aha2-6 root cells was remarkably
181 reduced compared with wild type (Figure 3E). The H+ efflux rates in bak1 root cells were also
182 reduced, although the reduction in bak1 mutants was weaker than that in aha2-6 mutant (Figure
183 3E). In addition, pH dye (bromocresol purple) was used to test the AHA2 activity in different
184 mutants (Yang et al., 2019; Cao et al., 2020). The results showed that the acidification around
7
185 bak1-4 and aha2-6 roots was impaired under LK conditions compared to wild type (Figure 3F).
186 All these data indicate that BAK1 may regulate the H+ efflux activity of AHA2, subsequently
187 affecting AKT1- and HAK5-mediated K+ uptake under LK conditions.
188
189 SERK1 is also involved in LK response

190 BAK1/SERK3 belongs to the SERK family, and the members in this family have functional
191 redundancy (Liu et al., 2020). Here, we observed the phenotypes of serk mutants to test their
192 functional redundancy in LK signaling. As shown in Figure 4, serk1, serk2, and serk4 single
193 mutants did not exhibit leaf chlorosis phenotype. However, serk1-1 bak1-4 double mutant
194 displayed an enhanced sensitive phenotype compared with bak1-4 single mutant under LK
195 conditions (Figure 4A), suggesting that SERK1 also participates in LK response. A previous
196 study found that the homozygous bak1-4 serk4-1 double mutant was lethal (He et al., 2007). In
197 this study, the heterozygous double mutant bak1-4+/- serk4-1 did not show obvious LK sensitive
198 phenotype (Figure 4C). In addition, neither the heterozygous triple mutant serk1-1 bak1-4+/-
199 serk4-1 nor serk2-1 bak1-4+/- serk4-1 displayed LK sensitive phenotype (Figure 4D and 4E),
200 suggesting that only homozygous mutation of BAK1 gene leads to LK sensitive phenotype.
201 According to the phenotypes of these mutants, it seems that BAK1 plays a major role in
202 regulating AHA2 in LK signaling, besides SERK1 is also involved in LK response. Since serk1-1
203 bak1-4 double mutant exhibited more severe defective phenotype than aha2-6 mutant (Figure
204 4A), SERK1 may regulate some other components in LK signaling pathway.
205
206 BAK1 interacts with the C terminus of AHA2

207 Previous reports have revealed that the activity of AHA2 can be regulated by protein kinases
208 (Fuglsang et al., 1999; Maudoux et al., 2000; Haruta et al., 2014; Fuglsang et al., 2014; Fuglsang
209 et al., 2007). Considering BAK1 and AHA2 may work together, we assumed that BAK1 could
210 directly interact with and phosphorylate AHA2 to regulate its activity. The results of both
211 split-LUC assays and yeast-two hybrid assays indicated that BAK1 directly interacted with
8
212 AHA2 (Figure 5A, S3G and 5B). The protein interaction between CIPK23 and AKT1 (Xu et al.,
213 2006) was used as positive control in split-LUC assays (Figure 5A). The interaction between
214 BAK1 and BIK1 (Lu et al., 2010; Lin et al., 2014) as well as the interaction between PKS5 and
215 AHA2 (Fuglsang et al., 2007) were used as positive controls in yeast-two hybrid assays (Figure
216 5B). Co-IP assays were also carried out to confirm protein interaction. Total membrane proteins
217 were extracted from the roots of Col/pSuper:AHA2-MYC transgenic line and wild type, then
218 immunoprecipitated with anti-MYC antibody. The presence of BAK1 and AHA2-MYC in the
219 immunoprecipitation fraction indicated their interaction in vivo (Figure 5C). Many studies have
220 shown that the C-terminus (850-948 aa) of AHA2 is the major domain regulated by protein
221 kinases (Falhof et al., 2016; Haruta et al., 2015; Rudashevskaya et al., 2012). In this study, the
222 C-terminus of AHA2 (AHA2 C-MYC) together with BAK1-FLAG was co-transformed into
223 Arabidopsis protoplasts for Co-IP assays. The results confirmed that BAK1 indeed interacted
224 with AHA2 C-terminus (Figure 5D).
225 It is known that the C-terminus of AHA2 contains self-inhibition region. When the
226 C-terminus is removed, the activity of AHA2 is increased (Regenberg et al., 1995;
227 Rudashevskaya et al., 2012). Therefore, the construct AHA2Δ98 (deletion of 851-948 aa) driven
228 by a β-estradiol (E2) inducible promoter (Schlücking et al., 2013) was transformed into bak1-4
229 mutant. As shown in Figure 5E, without E2 in the LK medium, the transgenic line
230 bak1-4/AHA2Δ98 exhibited a sensitive phenotype similar to bak1-4 mutant. Upon application of
231 E2 (10 μM) in the LK medium, the sensitive phenotype of transgenic line was restored (Figure
232 5E to 5G), suggesting that the constitutively activated AHA2 could complement the defective
233 phenotype of bak1 mutant. These results further confirm that BAK1 interacts with AHA2
234 C-terminus and regulates its activity, by which BAK1 affects K+ uptake under LK conditions.
235
236 BAK1 phosphorylates AHA2 at T858, T881, S944, Y946, and T947 in vitro
237 Phosphorylation is one of the important mechanisms that regulates AHA2 activity. The
238 phosphorylation sites are mainly located in the C-terminus of AHA2. To verify whether BAK1
9
239 could phosphorylate AHA2, we purified the N-terminus (AHA21-60 aa), the center loop
240 (AHA2299-644 aa), and the C-terminus (AHA2850-948 aa) of AHA2, which were used for in vitro
241 phosphorylation assays. The results showed that BAK1 mainly phosphorylated the C-terminus of
242 AHA2 (Figure 6A). To identify the phosphorylation sites, the C-terminus of AHA2 was divided
243 into five segments (C-1 to C-5, Figure 6B and 6G), and each of them contained several possible
244 phosphorylation sites. BAK1 mainly phosphorylated C-1 and C-5 segments (Figure 6B) that
245 contained three and four potential phosphorylation sites, respectively. Then, these sites were
246 mutated to alanine (A) to identify which sites were phosphorylated by BAK1. The results
247 indicated that BAK1 phosphorylated AHA2 at T858, T881, S944, Y946, and T947 in vitro
248 (Figure 6C to 6F).
249
250 Phosphorylation of T858 and T881 play important roles in LK response in vivo
251 In order to validate the candidate phosphorylation sites in vivo, we purified root membrane
252 proteins of wild type and bak1-4 mutant grown on LK and HK medium, then performed
253 quantitative phosphoproteomic assays to identify phosphorylation sites and compare their
254 phosphorylation levels. Among the five candidate sites, only T881 and T947 were detected to be
255 phosphorylated in vivo (Figure 6H, 6I and Table S1). The phosphorylation level of T881 was
256 increased under LK condition compared to HK in wild-type plants, while in bak1-4 mutant the
257 phosphorylation level was substantially reduced compared to the wild type, and not induced by
258 LK treatment (Figure 6H). These data confirm that BAK1 phosphorylates T881 in vivo under LK
259 condition. Comparatively, the phosphorylation level of T947 was neither increased under LK
260 condition, nor reduced in bak1-4 mutant (Figure 6I). Both S944 and Y946 were not
261 phosphorylated in vivo (Table S1), while T858 sites was not detected.
262 In addition, we also tested the functions of T858, T881, S944, Y946, and T947 in yeast.
263 These sites were mutated to dephosphorylated forms (A) or continuous phosphorylated forms
264 (D/E) one by one. Then, the H+ pump activities of mutated AHA2 were detected in RS-72 yeast
265 strain (Cid et al., 1987; Fuglsang et al., 2007). The results showed that S944, Y946, and T947
10
266 had little effect on AHA2 activity (Figure S5A), while T881D partially increased AHA2 activity
267 (Figure 7A). Although the phosphorylation status of T858 was not determined in
268 phosphoproteomic assays, both T858D and T858E substantially enhanced the activity of AHA2
269 in yeast (Figure 7A). These data suggest that both T858 and T881 may play roles in regulating
270 AHA2 activity.
271 To further investigate the functions of T858 and T881 in planta, the constructs AHA2T858D
272 and AHA2T881D driven by AHA2 native promoter were transformed into aha2-6 and bak1-4
273 mutants, respectively, then the LK phenotypes of transgenic plants were observed. The results
274 indicated that both AHA2T858D and AHA2T881D could partially complement the leaf chlorosis
275 phenotypes of aha2-6 and bak1-4 mutants under LK conditions (Figure 7B to 7I). All these data
276 demonstrate that BAK1 phosphorylates AHA2 at T858 and T881 to strengthen transmembrane
277 potential and H+ gradient in root cells, which subsequently promotes K+ uptake under LK
278 conditions.
279
280 BAK1 does not regulate HAK5 transcription through SGN3-LKS4 signaling pathway
281 Our recent study has revealed that LRR-RLK SGN3 interacts with RLCK (receptor-like
282 cytoplasmic kinase) LKS4/SGN1 forming a receptor kinase complex (Wang et al., 2021). Then,
283 LKS4 phosphorylates RBOHC and RBOHD to produce ROS signal, and ultimately promotes the
284 expression of HAK5 under LK stress (Wang et al., 2021). To verify the relationship between
285 BAK1-AHA2 pathway and SGN3-LKS4 pathway, we compared the LK phenotypes of their
286 mutants. The LK sensitive phenotype of sgn3 mutant was more severe than that of bak1-4 and
287 aha2-6 single mutants (Figure 8A and 8B). In addition, the bak1-4 lks4 double mutant showed an
288 enhanced leaf chlorosis phenotype compared to their single mutants (Figure 8C). RT-qPCR
289 analyses showed that the LK-induced HAK5 transcription was impaired in sgn3 and lks4 mutants,
290 but was not affected in bak1 mutant (Figure 8D). These data demonstrate that the two RLKs
291 BAK1 and LKS4 are involved in different LK signaling pathways.
292
11
12
293
294 DISCUSSION
295 BAK1, also known as SERK3, belongs to SERK family that contains five members (SERK1-5).
296 SERK5 is a non-functional kinase in Col-0 ecotype (Aan den Toorn et al., 2015; He et al., 2007;
297 Wu et al., 2015), therefore, studies on SERK proteins mainly focus on SERK1 to SERK4.
298 Previous reports showed that SERK family members regulate diverse physiological processes
299 and have functional redundancy (Liu et al., 2020). For example, SERK1, BAK1, and SERK4
300 regulate brassinosteroid (BR) signaling pathway (Gou et al., 2012). BAK1 and SERK4 function
301 in plant immunity and cell death containment (Roux et al., 2011; He et al., 2007). SERK1,
302 SERK2, BAK1, and SERK4 are all involved in the regulation of stomatal pattern (Meng et al.,
303 2015). In this study, phenotype comparison demonstrated that BAK1 plays a primary role in
304 regulating AHA2 in LK signaling (Figure 4). The serk1-1 bak1-4 double mutant displayed an
305 enhanced sensitive phenotype compared with bak1-4 single mutant (Figure 4A), suggesting that
306 SERK1 is also involved in LK response. Considering that serk1-1 bak1-4 showed a more severe
307 defective phenotype than aha2-6 mutant (Figure 4A), SERK1 may regulate some other
308 components in LK signaling pathway.
309 Both K+ uptake mutants (such as akt1 and hak5) and K+ translocation mutants (such as
310 nrt1.5) showed reduced shoot K+ content that led to leaf chlorosis phenotypes under LK
311 conditions (Xu et al., 2006; Li et al., 2017; Du et al., 2019; Wang et al., 2021; Figure S2B, S2C
312 and S2D). However, only nrt1.5 exhibited increased root K+ content (Figure S2D), because K+
313 cannot be transported to shoots resulting in K+ accumulation in roots (Li et al., 2017; Du et al.,
314 2019). This K+ distribution pattern (higher in root but lower in shoot) was not present in bak1
315 mutant (Figure S2D). SKOR is an outward K+ channel that also mediates root-to-shoot K+
316 translocation (Gaymard et al., 1998). Although the K+ concentration in skor xylem sap was
317 reduced (Han et al., 2016), skor mutant neither showed obvious sensitive phenotype nor reduced
318 K+ content under LK conditions (Figure S2; Li et al., 2017). The K+ depletion experiments
319 indicated that the root K+ uptake rates of bak1 mutants were substantially reduced (Figure 2B).
320 All these results demonstrate that BAK1 mainly regulates K+ uptake in LK response. However,
13
321 BAK1 and AHA2 also operate in vascular tissues (Grunwald et al., 2021), so it is possible that
322 BAK1-AHA2 may also contribute to root-to-shoot K+ transport under LK conditions.
323 A number of protein kinases have been characterized to phosphorylate AHA2 C-terminus
324 and modulate its activity. So far, many different phosphorylation sites at C-terminus have been
325 identified and show different effects. Some of them enhance AHA2 activity, while others reduce
326 its activity, forming a flexible and complex regulatory system (Rudashevskaya et al., 2012;
327 Falhof et al., 2016). In this study, we found that BAK1 phosphorylated T858 and T881 to
328 regulate AHA2 activity (Figure 6 and 7). T881 has been reported as an important
329 phosphorylation site before (Fuglsang et al., 2014), and our data suggest that T858 is also a
330 phosphorylation site. Functional characterization in yeast and plants demonstrated that both T858
331 and T881 play roles in regulating AHA2 activity in LK response (Figure 7). In our
332 phosphoproteomic data, the peptide “T858AFTMK” was not detected. This peptide was too short
333 after trypsin enzymolysis, besides T858 located at the edge of this peptide. Therefore, it was
334 difficult to determine the phosphorylation status of T858 using phosphoproteomic methods.
335 Phenotype comparison showed that AHA2T858D and AHA2T881D only partially complemented
336 the leaf chlorosis phenotype of aha2 and bak1 mutants (Figure 7B to 7I). When we constructed
337 the complementation lines, the AHA2 CDS sequence driven by its native promoter (2.3 kb) was
338 used. However, we found that the expression of AHA2 gene did not return to wild-type level in
339 these transgenic lines (Figure S5B). The low expression levels of AHA2 led to the partial
340 complementation phenotypes.
341 Previous studies have revealed that BAK1 acts as a co-receptor to transmit signals in
342 diverse signaling pathways (Liu et al., 2020; Ma et al., 2016). When signal ligands, receptor
343 kinases, and BAK1 form complexes, the receptor kinases and BAK1 can phosphorylate each
344 other leading to downstream phosphorylation reactions, gene expression and other events, by
345 which external signals can be transmitted into cells. BAK1 is involved in both BR signaling
346 pathway and immune signaling pathway that have been widely investigated (Li et al., 2002; Nam
347 and Li, 2002; Chinchilla et al., 2007; Heese et al., 2007; Belkhadir et al, 2014; Kim and
14
348 Russinova, 2020). In this study, to investigate whether BAK1-involved LK response is related to
349 BR or immune signaling pathways, we tested the phenotypes of BR-related (det2, dwf4, bri1-301,
350 and bzr1-1D) mutants and immune-related (fls2 and bik1) mutants. However, none of these
351 mutants showed obvious leaf chlorosis phenotype under LK conditions (Figure S6). The bak1-5
352 mutant, containing a C408Y mutation, is defective in FLS2-dependent immune signaling but not
353 in BR signaling (Schwessinger et al., 2011). Phenotype test indicated that bak1-5 mutant also
354 showed leaf chlorosis phenotype similar to bak1-3 and bak1-4 under LK conditions (Figure S1C).
355 In addition, previous study reported that the receptor kinase BRI1 could directly interact with
356 AHA2 (Yuan et al., 2018). Here, we also detected the protein interaction between AHA2 and the
357 receptor kinase FLS2 (Figure S3J). However, bri1 and fls2 did not display obvious LK sensitive
358 phenotype similar to aha2 mutant (Figure S6A and S6B). These results suggest that
359 BAK1-involved LK signaling pathway is independent of BR and immune signaling pathways.
360 BAK1 can interact with many RLKs involving various signaling pathways. In this study, we
361 collected the mutants of these RLK genes to examine their phenotypes under LK conditions. As
362 shown in Figure S7, most of these mutants (including pskr1, pskr2, pepr1, herk2, hsl2, ghr1, efr,
363 and hae) did not show different phenotype compared to wild type. It has been reported that
364 AHA1 and AHA2 together with cation channel CNGC17 form a functional cation-translocating
365 unit that is activated by RLKs PSKR1 and BAK1 (Ladwig et al., 2015). Here, we found that
366 neither pskr1 nor cngc17 mutant exhibited LK sensitive phenotype similar to bak1 or aha2
367 mutant (Figure S4B and S7), suggesting that PSKR1 and CNGC17 are not involved in
368 BAK1-mediated LK response.
369 Our recent study revealed that the SGN3-LKS4 kinase complex phosphorylates RBOHC
370 and RBOHD to produce ROS signal that promotes HAK5 expression and root K+ uptake under
371 LK stress (Wang et al., 2021). By comparing phenotypes and HAK5 expression levels, we
372 demonstrate that LKS4 and BAK1 work in different LK signaling pathways. We noticed that the
373 RLK mutant sgn3/gso1 showed a leaf chlorosis phenotype that was more severe than lks4, bak1,
374 and aha2 single mutants (Figure 8A and 8B; Wang et al., 2021). LKS4 is specifically expressed
15
375 in root endodermis (Alassimone et al., 2016; Wang et al., 2021), while SGN3 and BAK1 show
376 much broader tissue expression patterns (Alassimone et al., 2016; Li et al., 2002; Nam and Li,
377 2002). A recent study has reported that SGN3 can also interact with BAK1 and SERK1 (Okuda
378 et al., 2020). We speculate that SGN3 may regulate both BAK1 and LKS4, by which SGN3 is
379 involved in two parallel signaling pathways.
380 Taken together, in this study we identify a K+ uptake signaling pathway that is regulated by
381 RLKs in LK response. When plants are subjected to LK stress, the LK signal could be somehow
382 received by an unknown LRR-RLK located at the PM of root cells. This kinase may interact with
383 BAK1 forming kinase complex that phosphorylates and activates AHA2. Thus, the increased H+
384 gradient across the PM and hyperpolarized membrane potential promote HAK5- and
385 AKT1-mediated K+ uptake, which help plants resist LK stress (Figure 9). However, the receptor
386 (LRR-RLK) that interacts with BAK1 in this pathway is still unknown, which needs to be
387 identified in the future.
388
16
389 MATERIALS AND METHODS
390 Plant Materials
391 The Arabidopsis (Arabidopsis thaliana) Columbia ecotype (Col-0) was used as wild type in this
392 study. The T-DNA insertion plants bak1-3 (SALK_034523) and the bak1-4 (SALK_116202),
393 aha2-4 (SALK_082786), aha2-5 (SALK_022010), aha2-6 (SALK_062371), aha1-6
394 (SALK_016325), aha1-7 (SALK_065288), lks4 (SALK_055095), sgn3 (SALK_064029), pskr1
395 (SALK_071659), efr (SALK_068675), hae (SALK_021905), pepr1 (SALK_060002), pskr2
396 (SALK_140876), hsl2 (SALK_057117), herk2 (SALK_105055), cngc17-1 (SALK_013813),
397 cngc17-2 (SALK_076540) were obtained from Eurasian Arabidopsis Stock Centre (uNASC).
398 nrt1.5 (SALK_043036) and skor (SALK_132944) were obtained from the ABRC. The serk
399 single and multiple mutants including serk1-1 (SALK_044330), serk2-1 (SALK_058020),
400 serk4-1 (SALK_057955), serk1-1 bak1-4, serk2-1 bak1-4, bak1-4+/- serk4-1, serk1-1 bak1-4+/-
401 serk4-1, serk2-1 bak1-4+/- serk4-1, and bak1-5 (contains a G2929A substitution that causes a
402 C408Y change in amino acid sequence) were obtained as described previously (Meng et al.,
403 2015). akt1 (SALK_071803) and hak5 (SALK_005604) were obtained as described previously
404 (Xu et al., 2006).
405 aha2-6 bak1-5 was generated by crossing aha2-6 with bak1-5. The BAK1 genomic
406 sequence was cloned into the pCAMBIA1300 vector (Cambia) driven by its native promoter (2.3
407 kb), and this construct was transformed into the bak1-3 mutant to obtain two complementation
408 lines (COM1 and COM2). In order to obtain the AHA2T858D and AHA2T881D transgenic plants,
409 AHA2T858D and AHA2T881D CDSs driven by AHA2 native promoter (2.3 kb) were cloned into the
410 pCAMBIA1300 vector (Cambia) and then the constructs were transformed into bak1-4 and
411 aha2-6, respectively. AHA2Δ98 (the constitutive activation form of AHA2) driven by β-estradiol
412 (E2) induced promoter (Schlücking et al., 2013) was transformed into bak1-4 to acquire the
413 bak1-4/AHA2Δ98 transgenic plants.
414 Arabidopsis transformation with Agrobacterium tumefaciens (strain GV3101) was carried
415 out by the floral dip method (Clough and Bent, 1998).
17
416
417 Phenotypic Analyses and Growth Conditions

418 For phenotype test, the medium contained 1.5 mM MgSO4, 1.25 mM NH4H2PO4, 2.99 mM
419 Ca(NO3)2. The actual and final K+ concentration in the K+-sufficient medium (HK) and low-K+
420 medium (LK) were adjusted to 5 mM and 80 μM, respectively, by adding KCl. For the
421 phenotypic analyses using different concentrations of K+, medium were also supplemented with
422 KCl. The MS medium contained 1.5 mM MgSO4, 2.99 mM CaCl2, 20.6 mM NH4NO3, 18.79
423 mM KNO3, and 1.25 mM KH2PO4. The microelements in LK, HK and MS medium were
424 consistent and all contained 0.1 mM MnSO4, 0.005 mM KI, 0.0001 mM CuSO4, 0.1 mM H3BO3,
425 0.0001 mM CoCl2, 0.03 mM ZnSO4, 0.001 mM Na2MoO4, 0.1 mM FeSO4, and 0.1 mM
426 Na2EDTA. All the medium used in this study contained 0.9% (w/v) agar (Ourchem) and 3% (w/v)
427 sucrose. Seeds were surface sterilized using 6% (v/v) NaClO and incubated at 4°C in darkness
428 for 3 d. Then, the seeds were germinated on LK or HK medium at 22°C under constant
429 illumination (60-80 μmol m–2 s–1). Each phenotype test was performed at least three times. The
430 fresh shoots of 12-day-old seedlings grown on LK or HK medium were harvested for chlorophyll
431 content measurement. The chlorophyll was extracted in 80% acetone (v/v) in the dark for 1-2
432 days. Then, the absorbance of the extraction buffer at 645 and 663 nm was measured.
433 For seed harvesting, Arabidopsis plants were cultured in the potting soil mixture (rich
434 soil:vermiculite = 2:1, v/v) and kept in growth chambers (temperature was 22°C, illumination
435 was 120 μmol m-2 s-1 and the relative humidity was approximately 70%) with long-day
436 conditions (16 h light/8 h darkness).
437
438 K+ Content Measurement

439 The Arabidopsis seeds were germinated on low K+ and high K+ for 10 d, and then the seedlings
440 were used for K+ content measurement. The shoots and roots were harvested separately. For low
441 K+ treatment, 80 to 120 individual seedlings were collected as one biological replicate. For K+
442 sufficient treatment, 40 to 50 individual seedlings were collected as one biological replicate.
18
443 Three or four independent biological replicates were used in one experiment.
444 The collected samples were dried at 80°C to a constant weight and then the dry weight was
445 measured. The samples were treated in a muffle furnace at 300 °C for 1 h, and then 575°C for 5 h.
446 The ashes were dissolved and diluted in 0.1 N HCl. The K+ concentrations were measured using
447 the 4100-MP AES device (Agilent).
448
449 Transcription Analyses

450 The roots of 7-day-old seedlings were collected and used for total RNA extraction by using the
451 Trizol reagent (Invitrogen). For the LK induction experiment in Figure 8D, 5-day-old seedlings
452 were transferred to LK or HK medium for 1 day. Then the roots of 200-250 individual seedlings
453 were collected and used as one sample. Three or four independent samples (“n” indicated in the
454 figure legends) were used in one experiment. Three independent experiments were performed.
455 The total RNA was treated with DNase I (RNase Free, Takara) for 37°C 30 min, and then 65°C
456 for 10 min to eliminate DNA contamination. The complementary DNA (cDNA) was synthesized
457 by RevertAid First Strand cDNA Synthesis Kit (Thermo).
458 For RT-qPCR, the cDNA was diluted 40-fold with double-distilled water, and 8 μL of
459 diluted cDNA was used as the template in each reaction. 20 μL was one reaction volume
460 contained 10 μL SYBR Green mix (2×M5 HiPer Realtime PCR Super Mix with Low Rox, Mei5
461 Biotechnology), 8 μL cDNA, 2 μL forward and reverse primers (1 μM) and reacted on a 7500
462 Real Time PCR System machine (Applied Biosystems). The PCR was conducted as follows:
463 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. To normalize the
464 test gene expression levels, ACTIN2/8 was used as an internal standard. The primers were shown
465 in Supplemental Table S2.
466
467 Yeast Two-hybrid Assays

468 The vector construction refers to the Dual membrane kit user manual (Dualsystems Biotech).
469 Different empty vectors were selected according to the properties of the protein. BAK1 was
19
470 cloned into pBT3-SUC and AHA2 was constructed into pPR3-STE. The vector was transformed
471 into yeast strain NMY51 by PEG transformation method. Each transformation system contains
472 240 μL 50% PEG (w/v), 36 μL 1 M CH3COOLi, 25 μL 2 mg/mL ssDNA, 1.5 μg plasmid and
473 100 μL yeast cell solution (50 mL yeast culture medium with OD600 between 0.6 and 0.8 was
474 collected and resuspended with 2.5 mL ddH2O). The transformed strains were cultured on
475 SD/-Trp-Leu plates and confirmed by PCR. These transformants were then grown on
476 SD/-Trp-Leu-His-Ade plates for interaction detection.
477
478 Split-Luciferase Complementation Assays

479 BAK1 CDS was cloned into pCAMBIA1300-nLUC and pCAMBIA1300-cLUC to generate
480 BAK1-nLUC and BAK1 cLUC. AHA2 CDS was also cloned into pCAMBIA1300-nLUC and
481 pCAMBIA1300-cLUC to generate AHA2-nLUC and AHA2 cLUC. The constructed plasmids
482 were transformed into Agrobacterium (strain GV3101) and infiltrated into Nicotiana
483 benthamiana leaves for 3 d. LUC signal was collected by using a cooled CCD camera
484 (iKon-L936; Andor Tech) after spraying 1 mM of D-luciferin on the leaves (Promega; Chen et al.,
485 2008). The luminescence intensity was measured by the Image J program.
486
487 Co-IP (Co-immunoprecipitation)

488 In vitro Co-IP: The AHA2850-948 aa CDS was amplified and cloned into pSUPER1300-MYC
489 plasmid to create AHA2850-948 aa -MYC construct. The BAK1 CDS and PKS5 CDS were
490 amplified and cloned into pSUPER1300-FLAG plasmid to create BAK1-FLAG and
491 PKS5-FLAG constructs, respectively. The Arabidopsis leaf protoplasts of Col-0 were isolated
492 and transformed with the constructed plasmids, then grown at 22°C condition in darkness for 14
493 h. Total proteins from the protoplasts were isolated with the extraction buffer which contains 10
494 mM Tris-HCl (pH7.6), 150 mM NaCl, 2 mM EDTA, 0.5% NP-40 (v/v), 1 mM PMSF and 1×
495 protease inhibitor cocktail (Roche). Anti-MYC tag conjugated agarose beads (Sigma,
496 A7470-1ML) were used for immunoprecipitation, and the immunoprecipitates were examined by
20
497 western-blot using anti-MYC (Abmart, M20002L) and anti-FLAG (MBL, M185-3L) antibodies,
498 respectively.
499 In vivo Co-IP: The AHA2 CDS was amplified and cloned into pSUPER1300-MYC plasmid
500 to create AHA2-MYC construct and the Col/pSuper:AHA2-MYC transgenic lines were
501 constructed by transforming pSuper:AHA2-MYC vector into Col-0. Col and
502 Col/pSuper:AHA2-MYC transgenic lines were germinated at high K+ medium for 5 d and then
503 the seedlings were transferred to high K+ medium and low K+ medium for 1 d. The roots of
504 1200~1500 individual seedlings were collected for Co-IP. The total membrane proteins were
505 extracted by using the MinuteTM Total and Plasma Membrane Isolation Kit (Invent, SM-005).
506 Anti-MYC tag conjugated agarose beads (Sigma, A7470-1ML) were used for
507 immunoprecipitation, and the immunoprecipitates were examined by western-blot using
508 anti-MYC (Abmart, M20002L) and anti-BAK1 antibodies, respectively.
509
510 Yeast Complementation Assays

511 Saccharomyces cerevisiae strain RS-72 (Cid et al., 1987; Fuglsang et al., 2007) was used for
512 complementation tests. In RS-72 yeast, the natural constitutive promoter of the endogenous
513 plasma membrane H+-ATPase PMA1 was replaced by GAL1 (galactose-dependent promoter). A
514 yeast expression vector pMP1745 was used for expression of AHA2 under the control of PMA1
515 promoter. To generate the various point mutations of AHA2, the QuikChange Lightning Site
516 Directed Mutagenesis Kit (Agilent Technologies, 210518-5) was used to conduct point mutation
517 PCR, and AHA2-pMP1745 was used as the template. The reaction volume and PCR program
518 referred to the manufacturer’s instruction. The activity of AHA2 can be reflected by the growth
519 of RS-72 transformed with AHA2 or AHA2 with various point mutations on the SD/-Leu
520 medium with glucose as carbon source.
521
522 Phosphorylation Assays

523 The BAK1-C (748 bp to 1848 bp) was cloned into the pET-30a (+) vector (Novagen) to obtain
21
524 the protein expression plasmid His-BAK1-C. The AHA2-C (2548 bp to 2844 bp) was cloned into
525 the pGEX4T-1 vector (Pharmacia) to obtain the protein expression plasmid GST-AHA2-C.
526 His-BAK1-C and GST-AHA2-C were expressed in E. coli strain BL21 and purified using
527 Ni-Sepharose 6 Fast Flow (GE Healthcare) and Glutathione sepharose beads (GE Healthcare),
528 respectively. Site mutagenesis of GST-AHA2-C was performed with a QuikChange Lightning
529 Site Directed Mutagenesis Kit (Agilent Technologies, 210518-5).
530 For in vitro phosphorylation assays, purified kinase and substrate proteins were incubated in
531 phosphorylation reaction buffer containing 20 mM Tris-HCl (pH7.5), 10 mM MgCl2, 1 mM DTT,
532 1 μCi [γ-32P] ATP and 10 μM cold ATP. The reaction was reacted at 30 °C for 30 min, then the
533 SDS loading buffer was added and the mixture was treated at 95 °C for 5min to stop the
534 reactions. SDS-PAGE was performed and phosphorylation signal detected on a Typhoon 9410
535 imager. Coomassie brilliant blue stains served as loading controls.
536 For phosphoproteomics analyses, total membrane proteins extracted from the roots of
537 7-day-old seedlings grown on LK or HK media were used. Before the enrichment of
538 phosphorylated peptides, proteomics experiments were performed to determine the contents of
539 various peptides. After the enrichment of phosphorylated peptides, the phosphorylated peptides
540 were detected. Phosphorylation peak area represented the intensity of phosphorylation and the
541 ratio of phosphorylation peak area to corresponding peptide content was calculated. The relative
542 phosphorylation intensity is shown as indicated in the article.
543
544 Mensuration of H+ Flux and Root Acidification Assays

545 Seedlings germinated on high K+ medium for 5 days were transferred to low K+ medium for 3
546 days and were used for H+ fluxes measurement. The H+ fluxes were measured in the maturation
547 zone (the area where the root hairs begin to grow) using the Non-invasive Micro-test Technology,
548 (Younger; Xuyue [Beijing] Sci &Tech). The H+ selective micropipettes were filled with 50 μm
549 columns of H+-selective liquid exchanger and 1 cm columns of electrolyte buffer containing 15
550 mM NaCl and 40 mM KH2PO4 (pH 7.0). An Ag/AgCl wire electrode was inserted into the
22
551 micropipettes from the back such that it made contact with the electrolyte buffer. The H+
552 concentration was evaluated by moving the H+ microelectrode between two positions at 20 to 30
553 μm from the root. The H+ flux was measured in the testing solution (0.1 mM CaCl2, 0.1 mM KCl,
554 0.03 mM MES, pH 5.80) after the seedlings were balanced in the test solution for 10 min.
555 For root acidification assays, 7-day-old seedlings were transferred to LK or HK medium
556 containing 0.003% (w/v) bromocresol purple (pH 6.6) for 4 days.
557
558 Two-Electrode Voltage-Clamp Recordings from Xenopus laevis Oocytes

559 The CDSs of BAK1, AKT1, CIPK23, CBL1 were constructed to pGEMHE vector. The cRNA
560 was obtained in vitro using the mMESSAGE mMACHINE T7 kit (Ambion). Oocytes were
561 isolated from X. laevis and used to be injected with cRNA. Oocytes injected with water were
562 used as control and each oocyte was injected with 25 nL cRNA. The AKT1-expressing oocytes
563 were injected with a RNA mixture of AKT1, CIPK23, and CBL1 (3:1.5:1.5 ng in 25 nL). The
564 AKT1- and BAK1-coexpressing oocytes were injected with a RNA mixture of AKT1, CIPK23,
565 CBL1 and BAK1 (3:1.5:1.5:3 ng in 25 nL). Oocytes injected with cRNA were cultured in MBS
566 buffer (88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO3)2, 0.91 mM CaCl2, 0.82 mM MgSO4, 2.4
567 mM NaHCO3, 10 mM HEPES, 0.1 mg/mL streptomycin , 0.1 mg/mL gentamycin, pH7.5) for 36
568 h and used for current record.
569 A two-electrode voltage-clamp technique was applied to record K+ current using a
570 GeneClamp 500B amplifier (Axon Instruments) at room temperature (20ºC). K+ current was
571 recorded after oocytes were placed in K+ bath solution containing 96 mM KCl, 1.8 mM CaCl2,
572 1.8 mM MgCl2, 1 mM LaCl3, 10 mM HEPES (pH7.2, adjusted with NaOH/HCl). The
573 microelectrodes were filled with 3 M KCl. Whole-cell currents were filtered at 1 kHz and
574 digitized through a Digidata 1322A AC/DC converter using Clampex 9.0 software (Axon
575 Instruments).
576
577 Kinetic Analysis of K+ Uptake
23
578 0.6 g fresh weight of 7-day-old seedlings were collected as one sample and pretreated in a 80 mL
579 beaker (Figure S2A) containing one-quarter-strength HK solution (0.375 mM MgSO4, 0.31 mM
580 NH4H2PO4, 0.75 mM Ca(NO3)2, 5 mM KCl and 5 mM MES, pH 5.8 adjusted with Tris). After
581 growing overnight, the seedlings were transferred into 25 mL K starvation solution (200 μM
582 CaSO4, 5 mM MES, pH 5.8 adjusted with Tris) for 2 d. During these two days, K starvation
583 solution was changed three times a day. Then, the seedlings were transferred to 25 mL K
584 depletion solution (200 μM CaSO4, 250 μM KNO3, 5 mM MES, pH 5.8 adjusted with Tris) for 5
585 min, the solution samples were collected at different time points. All samples were shaken on a
586 shaking table at 22°C under constant illumination during the experiments (Xu et al., 2006). The
587 K+ concentrations were measured using the 4100-MP AES device (Agilent). The ratio of
588 potassium absorption to root fresh weight was calculated.
589
590 Statistical Analyses

591 Data are presented as the means ± SE from three or four separate samples (denoted by “n” in the
592 figure legends). For statistical significance, one-way analysis of variance test was performed,
593 with different lowercase letters indicating a significant difference at P<0.05.
594
595 Accession numbers

596 Sequence data for the genes described in this article can be found in the Arabidopsis TAIR
597 database (https://www.arabidopsis.org) under the following accession numbers: AT4G33430 for
598 BAK1, AT4G30190 for AHA2, AT2G26650 for AKT1, AT4G13420 for HAK5. AT2G18960 for
599 AHA1, AT2G39660 for BIK1, AT5G46330 for FLS2, AT2G30360 for PKS5, AT4G17615 for
600 CBL1, AT1G30270 for CIPK23.
601
602 Supplemental Data

603 Supplemental Figure S1. Phenotype test of bak1 mutants under LK conditions.
604 Supplemental Figure S2. Phenotype and K+ content of nrt1.5 and skor mutants.
24
605 Supplemental Figure S3. Analyses of the interaction and regulation between BAK1 and AKT1
606 or HAK5.
607 Supplemental Figure S4. Phenotype test of aha1 and cngc17 mutants.
608 Supplemental Figure S5. Effects of S944, Y946, and T947 on AHA2 activity.
609 Supplemental Figure S6. Phenotype test of BR-related and immune-related mutants.
610 Supplemental Figure S7. Phenotype test of RLK mutants.
611 Supplemental Table S1. The phosphorylation information of T881 and T947 in
612 phosphoproteomic data analyzed by MASCOT software.
613 Supplemental Table S2. Primer sequences used in this study.
614
615 Funding Information

616 This work was supported by grants from the National Key Research and Development Program
617 of China (2020YFA0509902), the National Natural Science Foundation of China (No. 32025004,
618 32161133014, and 31921001), and Beijing Outstanding University Discipline Program.
619
620 ACKNOWLEDGMENTS
621 We thank Dr. Libo Shan (Texas A&M University, USA) for providing serk single and multiple
622 mutants. We thank Jörg Kudla (Wilhelms-Universität Münster, Germany) for providing the
623 SUPERR:sXVE:GFPC:Bar vector. We thank Dr. Jianmin Zhou (Chinese Academy of Sciences,
624 China) for providing the fls2 and bik1 mutants. We also thank Dr. Tonglin Mao (China
625 Agricultural University) and Dr. Jia Li (Lanzhou University) for providing the det2, dwf4,
626 bri1-301 and bzr1-1D mutants.
627
628
25
629 FIGURE LEGENDS
630 Figure 1. bak1 mutants are sensitive to low K+ stress.
631 (A) Phenotypic comparison among wild-type plants (Col) and two bak1 mutants. Seedlings were
632 germinated and grown on low K+ medium (LK, 80 μM K+) or K+-sufficient medium (HK, 5 mM
633 K+) for 12 d.
634 (B) K+ content measurement of indicated plants shown in (A) after being germinated and grown
635 on LK or HK medium for 10 d. Data are presented as means ± SE (n = 4). DW refers to dry
636 weight. Different lowercase letters indicate a significant difference at P < 0.05 based on one-way
637 analysis of variance (ANOVA).
638 (C) Phenotype test of bak1 complementation lines bak1-3/pBAK1:BAK1 (COM1 and COM2).
639 (D) K+ content of various plants as indicated. Seedlings were germinated and grown on LK or
640 HK medium for 10 d. Data are presented as means ± SE (n = 3). DW refers to dry weight.
641 Different lowercase letters indicate a significant difference at P < 0.05 based on one-way analysis
642 of variance (ANOVA).
643
644 Figure 2. BAK1 is involved in the K+ uptake regulation

645 (A) Total K+ amount (per plant) of Col, bak1-3, and bak1-4. The total K+ amounts were
646 measured after the seedlings were germinated and grown on low K+ medium (LK, 80 μM K+) or
647 K+-sufficient medium (HK, 5 mM K+) for 10 d. Data are presented as means ± SE (n = 4).
650 (B) Comparison of K+ uptake rates by using K+-depletion method among Col, bak1-3, bak1-4
651 and akt1. Data are shown as means ± SE (n = 3). FW refers to the Fresh weight of roots.
654 (C) and (D) Phenotype test of various plants. Seedlings were germinated and grown on LK or
655 HK medium for 12 d.
26
656
657 Figure 3. BAK1 regulates AHA2 activity.

658 (A) to (C) Phenotype test of various mutants. Seedlings were germinated and grown on low K+
659 medium (LK, 80 μM K+) or K+-sufficient medium (HK, 5 mM K+) for 12 d.
660 (D) K+ content measurement of indicated plants shown in (C). Seedlings were germinated and
661 grown on LK or HK medium for 10 d. Data are shown as means ± SE (n = 4). DW refers to dry
662 weight. Different lowercase letters indicate a significant difference at P < 0.05 based on one-way
663 analysis of variance (ANOVA).
664 (E) H+ fluxes measurement of Col (n = 9), bak1-4 (n = 7), bak1-5 (n = 8) and aha2-6 (n = 8).
665 Seedlings germinated on HK medium for 5 d were transferred to LK medium for 3 d and were
666 used for H+ fluxes measurement. The H+ fluxes were measured in the maturation zone (the area
667 where the root hairs begin to grow). Line chart (left) and histogram (right) are shown here.
670 (F) Visualization of medium acidification around roots. Seven-day-old seedlings germinated on
671 HK medium were transferred to LK or HK medium (containing 0.003% bromocresol purple at
672 pH 6.6) for 4 days.
673
674 Figure 4. SERK1 may be also involved in LK response.

675 Phenotype test of the single (A to C), double (A to C), and triple (D and E) mutants of SERK
676 genes. Seedlings were germinated and grown on low K+ medium (LK, 80 μM K+) or
677 K+-sufficient medium (MS, 20 mM K+) for 12 d.
678
679 Figure 5. BAK1 interacts with the C-terminus of AHA2.

680 (A) Split-luciferase complementation assay was used to detect the interaction between BAK1
681 and AHA2. The co-expression of AKT1-cLUC and CIPK23-nLUC was used as a positive control.
682 The luciferase signals were detected after the indicated constructs were expressed in N.
27
683 benthamiana leaves for 3 d.
684 (B) Interaction of BAK1 with AHA2 in yeast. The yeast strain NMY51 was transformed with
685 BAK1-Cub and AHA2-Nub vectors. BAK1-Cub and BIK1-Nub, PKS5-Cub and AHA2-Nub
686 were used as positive control. Yeast growth was shown on SD/-Trp-Leu or SD/-Trp-Leu-His-Ade
687 medium.
688 (C) Co-IP assay showing the interaction of BAK1 proteins with AHA2 proteins in planta. The
689 5-day-old seedlings of Col/pSuper:AHA2-MYC transgenic line and Col were transferred to low
690 K+ medium (LK, 80 μM K+) or K+-sufficient medium (HK, 5 mM K+) for 1 d, then their roots
691 were collected and used for Co-IP assays. Total membrane proteins were extracted and
692 immunoprecipitated with an anti-MYC antibody, and detected with an anti-BAK1 antibody.
693 (D) Co-IP assay showing the interaction of BAK1 proteins with AHA2 C-terminus in
694 Arabidopsis protoplasts. The BAK1-FLAG and AHA2 C-MYC (AHA2850-948 aa-MYC) driven by
695 pSuper promoter were transformed into Arabidopsis protoplasts. Total proteins were extracted
696 from protoplasts and immunoprecipitated with an anti-MYC antibody, and detected with an
697 anti-FLAG antibody.
698 (E) Phenotype comparison among Col, bak1-4, and bak1-4/AHA2Δ98. AHA2Δ98 (constitutive
699 activation form of AHA2) was constructed into an β-estradiol (E2)-induced expression vector
700 and generated SUPERR:sXVE:AHA2Δ98. bak1-4/AHA2Δ98 was obtained by transforming
701 SUPERR:sXVE:AHA2Δ98 into bak1-4. Seedlings were germinated and grown on LK or HK
702 medium with or without 10 μM β-estradiol (E2) for 12 d.
703 (F) Chlorophyll contents of indicated plants shown in (E). Data are shown as means ± SE (n = 4).
704 FW refers to Fresh weight. Different lowercase letters indicate a significant difference at P < 0.05
705 based on one-way analysis of variance (ANOVA).
706 (G) RT-qPCR analyses of AHA2 expression in the indicated plants shown in (E). Data are shown
707 as means ± SE (n = 4). Different lowercase letters indicate a significant difference at P < 0.05
709
28
710 Figure 6. BAK1 phosphorylates AHA2 at T858, T881, S944, Y946, and T947.
711 (A) BAK1 phosphorylates AHA2 C-terminus in vitro. The purified recombinant GST-AHA2 C,
712 GST-AHA2 N, GST-AHA2299-644 aa were incubated in protein kinase buffer (contained [γ-32P]
713 ATP) with His-BAK1 C, and separated on 10% SDS-PAGE. The autoradiograph of the gel is
714 shown in the upper panel, and the coomassie brilliant blue (CBB)-stained proteins are shown in
715 the lower panel.
716 (B) Segmentation diagram of AHA2 C-terminus. The length of each fragment is shown in the
717 figure. The red stars indicate GST-AHA2 C-1 to GST-AHA2 C-5. The segments of AHA2
718 C-terminus are shown in (G).
719 (C) and (D) Phosphorylation assays for analyzing the residues T858, T861, and Y866 in
720 GST-AHA2 C-1 (C) T942, S944, Y946, and T947 in GST-AHA2 C-5 (D). GST-AHA2 C-1 and
721 GST-AHA2 C-5 are indicated by red arrows.
722 (E) and (F) Phosphorylation assays for analyzing the residues T858, T881, S944, Y946, and
723 T947. Red arrows indicate GST-AHA2 C. The numbers below bands represent gray analysis
724 results.
725 (G) Segmentation diagram of AHA2 C-terminus. The length of each fragment is shown in the
726 figure. “aa” stands for amino acids.
727 (H) and (I) Phosphorylation levels of T881 and T947 in Col and bak1-4 under HK and LK
728 conditions. Data are shown as means ± SE (n = 3). Different lowercase letters indicate a
729 significant difference at P < 0.05 based on one-way analysis of variance (ANOVA).
730
731 Figure 7. Phosphorylation of T858 and T881 play important roles in LK response.
732 (A) The AHA2 activity was detected after T858 or T881 were mutated to dephosphorylated
733 forms (A) or continuous phosphorylated forms (D/E). AHA2Δ98, the constitutive activation form
734 of AHA2, was used as a positive control. The endogenous PM H+-ATPase gene in RS-72 was
735 induced by galactose. When AHA2 was transferred into the RS-72 yeast strain, the growth of the
736 yeast depended on AHA2 activity on glucose medium. Photographs were taken after 3 to 5 d of
29
737 growth on the indicated medium. The initial OD600 was 0.8, equal volumes of fourfold serial
738 dilutions applied for each yeast strain.
739 (B) to (E) Phenotypic comparison among Col, bak1-4, aha2-6, and transgenic plants. Seedlings
740 were germinated and grown on low K+ medium (LK, 80 μM K+) or K+-sufficient medium (HK, 5
741 mM K+) for 12 d.
742 (F) to (I) Chlorophyll contents of indicated plants shown in (B) to (E). Data are shown as means
743 ± SE (n = 3). FW refers to Fresh weight. Different lowercase letters indicate a significant
744 difference at P < 0.05 based on one-way analysis of variance (ANOVA).
745
746 Figure 8. BAK1 and LKS4 are involved in different LK signaling pathways.
747 (A) Phenotypic comparison among Col, bak1-4, sgn3, and aha2-6. Seedlings were germinated
748 and grown on low K+ medium (LK, 80 μM K+) or K+-sufficient medium (HK, 5 mM K+) for 12
749 d.
750 (B) Chlorophyll contents of indicated plants shown in (A). Data are shown as means ± SE (n = 4).
751 FW refers to Fresh weight. Different lowercase letters indicate a significant difference at P < 0.05
753 (C) Phenotypic comparison among various mutants. Seedlings were germinated and grown on
754 LK (80 μM K+ or 160 μM K+) medium for 12 d.
755 (D) RT-qPCR analyses of HAK5 expression in the indicated plants. Data are shown as means ±
756 SE (n = 3). Different lowercase letters indicate a significant difference at P < 0.05 based on
757 one-way analysis of variance (ANOVA).
758
759 Figure 9. Working model of AHA2 phosphorylation regulation by BAK1 in Arabidopsis

760 response to low K+ stress.
761 When plants are subjected to LK stress, the LK signal could be somehow received by an
762 unknown LRR-RLK located at the PM of root cells. This kinase may interact with co-receptor
763 BAK1 forming kinase complex. Then, BAK1 phosphorylates and activates PM H+-ATPase
30
764 AHA2 to establish H+ gradient across the PM and hyperpolarized membrane potential that
765 promote HAK5- and AKT1-mediated K+ uptake, respectively. Therefore, the K+ uptake in root
766 cells are enhanced, which help plants to resist LK stress. ‘‘P’’ represents the phosphorylation
767 process.
768
31
769 References
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39
Figure 1
A C
Col bak1-3 bak1-4 Col bak1-3 bak1-4 Col bak1-3 COM1 COM2 Col bak1-3 COM1 COM2
LK HK LK HK
B Col bak1-3 bak1-4

D Col bak1-3 COM1 COM2
K+ Content (mmol g-1 DW)


0.5 1.6 0.5 1.8 a
LK a HK LK aaa HK
a 1.4 a a aaaa 1.6
0.4 a 0.4 1.4
a 1.2
1.0 1.2
0.3 a a a 0.3 a 1.0 a aaa
0.8 a a
ab
0.2 bb 0.2 b 0.8
0.6 0.6
0.4 0.4
0.1 0.1
0.2 0.2
0.0 0.0 0.0 0.0
Shoot Root Shoot Root Shoot Root Shoot Root
Figure 2
Col bak1-5
A B
K Absorption (μmol g FW)

25 bak1-4 akt1
Col bak1-3 bak1-4
60 600
-1
K+ Amount (nmol/plant)
K+ Amount (nmol/plant)
a a 20 a
a a
50 LK 500 HK
15 b
40 400
b
30 300 10
20 b b 200 c
a
b b 5
10 100 a a a
+
0 0 0
Shoot Root Shoot Root 0 2 4 6 8 10 12 14 16
Time (h)
C D
akt1 akt1
Col bak1-4 bak1-4 akt1 Col bak1-4 bak1-4 akt1 Col bak1-3 bak1-4 hak5 Col bak1-3 bak1-4 hak5
LK HK LK HK
Figure 3
akt1 aha2-6
A Col aha2-6 aha2-4 aha2-5 B Col bak1-4
hak5
akt1 hak5 aha2-6 C Col bak1-5
bak1-5
aha2-6
LK LK LK
HK HK HK
D Col bak1-5 aha2-6 bak1-5 aha2-6

E Col bak1-4 bak1-5 aha2-6
160
H Flux (pmol cm s )
160
H Flux (pmol cm s )
-2 -1
-2 -1
0.5 LK 2.0 aa HK 140 a
1.8 a 140
aa 120
0.4 ab 1.6 b 120 ab
b 1.4 100 bc
100
0.3 1.2 80
a 1.0 a 80
ab ab 60 c
0.2 0.8 b 60
bb b 0.6 40
0.1 0.4 40 20
+
0.2 20 0
0.0 0.0 0 100 200 300 400
Shoot Root Shoot Root
Time (s)
Col bak1-4 aha2-6 Col bak1-4 aha2-6
F
pH decrease
LK HK
Figure 4
serk2-1 bak1-4+/-
A Col serk1-1
bak1-4 bak1-4 serk1-1 aha2-6 B Col bak1-4 bak1-4 serk2-1 aha2-6
Caha2-6 Col bak1-4 serk4-1 serk4-1
LK LK LK
MS MS MS
serk1-1 serk2-1
D bak1-4+/-
E bak1-4+/-
Col bak1-4 serk4-1 aha2-6 Col bak1-4 serk4-1 aha2-6
LK MS LK MS
Figure 5
Input Anti-MYC IP
A C
AHA2-cLUC AKT1-cLUC
BAK1-nLUC CIPK23-nLUC
Anti-MYC AHA2-MYC
BAK1-nLUC AHA2-cLUC
cLUC nLUC
Anti-BAK1 BAK1
HK LK HK LK
SD/-WL SD/-WLHA
Input Anti-MYC IP
B BAK1-Cub D AHA2 C-MYC
AHA2-Nub + + + + + + + +
+ - - - + - - - FLAG
BAK1-Cub
- - + - - - + - PKS5-FLAG
BIK1-Nub
- - - + - - - + BAK1-FLAG
PKS5-Cub
AHA2-Nub Anti-MYC AHA2 C-MYC
AHA2-Nub
Cub BAK1-FLAG
Anti-FLAG PKS5-FLAG
BAK1-Cub
Nub
bak1-4/ bak1-4/
E Col bak1-4 AHA2Δ98 Col bak1-4 AHA2Δ98 F
Col bak1-4 bak1-4/AHA2Δ98
a a a
a a
0.8 a
Chlorophyll Content
a a
(mmol g FW)
a
0.6
-1
0.4 b
bc
c
0.2
0.0
LK LK+10 μM E2 LK LK+10 μM E2 HK HK+10 μM E2
G 2.5 b
AHA2 Relative Expression
2.0
1.5
ab
a a a a
1.0
0.5
0.0
HK HK+10 μM E2
+E2
Figure 6
A B C
BAK1 C
GST-AHA2 C
Autorad
GST-AHA2299-644aa
BAK1 C
GST-AHA2 C
GST-AHA2 N * * * * *
GST CBB
D E F
BAK1 C
1 0.83 0.46 0.45 0.51 0.16 1 0.83 0.69 0.03 0.14 0.18
Autorad
BAK1 C
GST-AHA2 C
GST CBB
G H Col bak1-4 I Col bak1-4

Relative Phosphorylation
Relative Phosphorylation
1.6 a 1.2
T881 a T947
GST-AHA2 C 850-948 aa 1.4 1.0
850-880 aa ab
GST-AHA2 C-1 1.2 ab
.8
Intensity
Intensity
GST-AHA2 C-2 871-903 aa 1.0 b b

901-920 aa c c
GST-AHA2 C-3 .8 .6
GST-AHA2 C-4 911-940 aa .6 .4
GST-AHA2 C-5 933-948 aa .4
.2 .2
0.0 0.0
HK LK HK LK
Figure 7
Galactose Glucose
pH 5.5 pH 3.5 pH 4.5 pH 5.5
A
AHA2
AHA2T858A
AHA2T858D
AHA2T858E
AHA2T881A
AHA2T881D
AHA2Δ98
Empty vector
OD600 0.8 0.2 0.05
aha2-6/ bak1-4/ aha2-6/ bak1-4/
B pAHA2:AHA2T858D C pAHA2:AHA2T858D D pAHA2:AHA2T881D E pAHA2:AHA2T881D
Col aha2-6 1 2 Col bak1-4 1 2 3 Col aha2-6 1 2 Col bak1-4 1 2
LK LK LK LK
HK HK HK HK
F G Col bak1-4
H I
Col aha2-6 bak1-4/pAHA2:AHA2
T858D
-1 Col aha2-6 Col bak1-4
aha2-6/pAHA2:AHA2T858D-1 bak1-4/pAHA2:AHA2
T858D
-2 aha2-6/pAHA2:AHA2T881D-1 bak1-4/pAHA2:AHA2T881D-1
T858D T858D T881D T881D
aha2-6/pAHA2:AHA2 -2 bak1-4/pAHA2:AHA2 -3 aha2-6/pAHA2:AHA2 -2 bak1-4/pAHA2:AHA2 -2
1.0 0.8 a 0.8 0.8 a aa
aa a a a aa
Chlorophyll Content
Chlorophyll Content
a aaa
Chlorophyll Content
Chlorophyll Content
a a
0.8
(mmol g-1 FW)
(mmol g-1 FW)
b
(mmol g-1 FW)
(mmol g-1 FW)
0.6 b 0.6 0.6

a ab ab b
b c
0.6 b c
c
0.4 0.4 0.4
0.4 bb d
0.2 d 0.2 0.2
0.2 d
c
0.0 0.0 0.0 0.0
LK HK LK HK LK HK LK HK
Figure 8
Col bak1-4 sgn3 aha2-6 Col bak1-4 sgn3 aha2-6
Col bak1-4
A B sgn3 aha2-6
0.7
a a aa a
Chlorophyll Content
0.6
(mmol g-1 FW)

0.5
0.4
0.3 b
0.2 c
0.1 d
0.0
LK HK
LK HK
lks4 lks4
C Col bak1-3 bak1-4 aha2-6 bak1-4 lks4 Col bak1-3 bak1-4 aha2-6 bak1-4 lks4 D Col bak1-4
lks4 sgn3
HAK5 Relative Expression

100
a
80 a
60
3.5
3.0
40 2.5
2.0
1.5
1.0
20 b b 0.5
0.0
0
80 μM K+ 160 μM K+ LK HK
CBL1/9
K+
AKT1
ADP+Pi
++++
----
Low K+ stress
H+ AHA2
H+
P
BAK1
ATP
?
CBL1/9
Extracellular
Figure 9
Cytoplasm
K + H+
HAK5
space
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Receptor-Like Protein Kinase Bak1 Promotes K Uptake by Regulating H - Atpase Aha2 Under Low Potassium Stress

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2 H+-ATPase AHA2 under low potassium stress

48 Keywords: Low potassium, receptor-like protein kinase, BAK1, AHA2, Arabidopsis

133 BAK1 is involved in the K+ uptake regulation

161 BAK1 may regulate AHA2 activity

189 SERK1 is also involved in LK response

206 BAK1 interacts with the C terminus of AHA2

417 Phenotypic Analyses and Growth Conditions

438 K+ Content Measurement

449 Transcription Analyses

467 Yeast Two-hybrid Assays

478 Split-Luciferase Complementation Assays

487 Co-IP (Co-immunoprecipitation)

510 Yeast Complementation Assays

522 Phosphorylation Assays

544 Mensuration of H+ Flux and Root Acidification Assays

558 Two-Electrode Voltage-Clamp Recordings from Xenopus laevis Oocytes

577 Kinetic Analysis of K+ Uptake

590 Statistical Analyses

595 Accession numbers

602 Supplemental Data

615 Funding Information

644 Figure 2. BAK1 is involved in the K+ uptake regulation

657 Figure 3. BAK1 regulates AHA2 activity.

674 Figure 4. SERK1 may be also involved in LK response.

679 Figure 5. BAK1 interacts with the C-terminus of AHA2.

759 Figure 9. Working model of AHA2 phosphorylation regulation by BAK1 in Arabidopsis

B Col bak1-3 bak1-4

K+ Content (mmol g-1 DW)

K+ Content (mmol g-1 DW)

K Absorption (μmol g FW)

D Col bak1-5 aha2-6 bak1-5 aha2-6

G H Col bak1-4 I Col bak1-4

GST-AHA2 C-2 871-903 aa 1.0 b b

(mmol g-1 FW)

0.6 b 0.6 0.6

(mmol g-1 FW)

HAK5 Relative Expression

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