Review

Tansley review Nudge-nudge, WNK-WNK (kinases), say no more?

Author for correspondence: Anh H. Cao-Pham1,2, Daisuke Urano3, Timothy J. Ross-Elliott1 and Alan M. Jones 1,4 Tel: +1 919 962 6932 Alan M. Jones Email: [email protected] 1Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; 2Department of Biological Received: 18 December 2017 Sciences, National University of Singapore, 117543, Singapore; 3Temasek Life Sciences Laboratory and Department of Biological Accepted: 1 May 2018 Sciences, National University of Singapore, 117604, Singapore; 4Department of Pharmacology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

Contents

Summary 35 V Functions of WNK family members in physiology and 41 development I Overview of animal and plant WNK kinases 35 VI. Say no more? Still many questions to be answered 45 II. Structure: domains and topology 36 Acknowledgements 46 III. Phylogeny–evolutionary relationships 41 References 46 IV. Plant WNK kinase distribution and regulation of WNK 41 expression and activity

Summary

New Phytologist (2018) 220: 35–48 WITH NO LYSINE (WNK) kinases are serine/threonine kinases uniquely characterized by an doi: 10.1111/nph.15276 anomalous placement of a catalytic lysine, hence their moniker. In animals, WNK protein kinases play critical roles in protein trafficking of components that mediate renal ion transport processes Key words: circadian, flowering, protein and regulate osmoregulation of cell volume. In plants, the WNK kinase gene family is larger and trafficking, regulator of G signaling, WNK more diverse. Recent studies revealed WNK kinase roles in orchestrating the trafficking of an ion kinase. channel, a lipid kinase complex in animals, and a heterotrimeric G protein signaling component in plants that is necessary for signal transduction. For this reason, new attention is geared toward investigating the mechanisms adopted by WNK kinases to nudge intracellular proteins to their destinations. In this review, the functions of WNK kinases in protein trafficking are derived from what we have learned from the model organism Arabidopsis thaliana. To place this new idea in context, we provide the predicted WNK kinase structures, their predicted expression patterns, a speculated evolutionary pathway, and the regulatory roles of plant WNKs in transport processes and other physiologies. We brazenly predict that the WNK kinases in both plants and animals will soon be recognized as a nexus for trafficking-based signal transduction.

I. Overview of animal and plant WNK kinases protein, serine/threonine kinase (MAPK) family in mammals. A defining characteristic of WNK family members is the absence of a Protein kinases belong to a superfamily of enzymes that modulate a catalytic lysine residue specifically located in the primary sequence wide spectrum of cellular processes through phosphorylating of a subdomain (subdomain II) within the N-terminal kinase downstream protein substrates. WITH NO LYSINE (WNK) domain. This lysine residue, conserved in all other kinases, is critical kinases are a class of protein kinase that was first discovered by Xu for coordination and binding ATP in the catalytic site. While also et al. (2000) in a screen for novel members of the mitogen-activated critical for catalysis, the relevant lysine residue in the WNK kinase

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subfamily is located at a different position in the kinase domain protein (AtRGS1) and mediates AtRGS1 endocytosis for sustained (specifically, subdomain I) (Xu et al., 2002). WNK kinases regulate activation of G-protein-coupled signaling (Fig. 1b). ion transporters like the Solute Carrier 12 (SLC12) family of Higher plant genomes encode more WNK kinases than animal cation–chloride cotransporters (CCCs) in mammals through a genomes do. The genetic model Arabidopsis has 11 WNK genes diverse range of catalytic-dependent mechanisms, thus controlling (AtWNK1 to AtWNK11) (Hong-Hermesdorf et al., 2006). All cell volume, transepithelial transport, and intracellular chloride Arabidopsis WNK genes, except for AtWNK6, are expressed in (Cl ) concentration (Fig. 1a) (Kahle et al., 2006). They also play a organs from the seedling to the flowering plant. Nakamichi et al. critical role in angiogenesis in zebrafish (Lai et al., 2014) and (2002) demonstrated that AtWNK1, AtWNK2, AtWNK4, and regulate Wnt signaling in Drosophila (Serysheva et al., 2013). As AtWNK6 might be influenced by the circadian clock and function will be discussed fully herein, plant WNK kinases also play various in a mechanism that generates circadian rhythms in Arabidopsis.V- roles in development and stress responses. ATPase is a potential substrate of AtWNK8 (Fig. 1b) (Hong- In humans, four different WNK genes were identified, namely Hermesdorf et al., 2006). WNK kinases regulate the floral WNK1, WNK2, WNK3, and WNK4 kinases (Verıssimo & Jordan, transition via the photoperiod pathway (Fig. 1b) (Wang et al., 2001; Wilson et al., 2001; Gamba, 2005). WNK kinases are 2008). To date, our clearest molecular understanding for the essential in renal physiology; gain-of-function mutations in WNK1 function of WNK kinases is in phosphorylation-dependent protein and WNK4 are implicated in pseudohypoaldosteronism type II, trafficking (Urano et al., 2012; Fu et al., 2014; Liao et al., 2017b). familial hyperkalemic hypertension (Wilson et al., 2001). Mam- malian WNK1, WNK3, and WNK4 are regulated by Cl , which II. Structure: domains and topology prevents activation of kinase properties by binding directly to the kinase catalytic site (Piala et al., 2014; Terker et al., 2016). 1. Kinase domain MAP4Ks STE20 (Sterile 20)-Like Kinases (SPAK) and Oxidative Stress Response Kinase-1 (OSR1) are the best studied substrates of Containing from 1243 to 2382 amino acids, human WNK kinases human WNK kinases (Fig. 1a). Phosphorylation by upstream are larger than Arabidopsis WNK kinases, which contain from 492 WNK kinases 1, 3, or 4 at the SPAK/OSR1 T-loop threonine of the to 701 amino acids for predicted molecular weights of 60–70 kDa catalytic domain activates SPAK and OSR1 (Vitari et al., 2005). (Xu et al., 2000; Verıssimo & Jordan, 2001; Wilson et al., 2001). Three different members of the SLC12 family of CCCs – Sodium– WNK kinases have 12 conserved subdomains comprising the Potassium-2-Chloride Cotransporters (NKCC1 and NKCC2), catalytic core of a kinase (Hanks & Hunter, 1995). The conserved and Sodium Chloride Cotransporter (NCC) – are then directly kinase domains of the four human WNK kinases have 85–90% in phosphorylated by activated SPAK/OSR1, creating a kinase amino acid sequence identity. However, for Arabidopsis WNKs, cascade (Fig. 1a) (Dowd & Forbush, 2003; Moriguchi et al., there is a wider variation in sequence similarities, ranging from 78 2005; Ponce-Coria et al., 2008; Richardson et al., 2008). Activa- to 96% (Wang et al., 2008). Multiple sequence alignment of tion of these transporters is essential in the regulation of cell volume AtWNK kinases affirms their identity through establishing the and intracellular Cl content under hyperosmotic or hypotonic existence of a well-conserved kinase domain that comprises 12 low Cl conditions (Kahle et al., 2006). Such a kinase cascade subdomains (subdomains I–XII) and resides toward the N terminal mediated by upstream WNKs may also exist in plants. For instance, half of the protein, as shown in Fig. 2. a study by Urano et al. (2012) suggested that plant Arabidopsis The following residue numbers are based on positions in human WNK8 phosphorylates Regulator of G protein Signaling (RGS) WNK1. In WNK kinases, the lysine that makes up the catalytic

Fig. 1 Overview illustration to compare between (a) animal WITH NO LYSINES (WNKs) and (b) Arabidopsis WNKs functions. (a) WNK kinases are best known to phosphorylate and activate STE20-RELATED PROLINE ALANINE-RICH KINASE (SPAK) and OXIDATIVE STRESS RESPONSE 1 (OSR1) kinases, which in turn phosphorylate various members of the SLC12 family of cation–chloride cotransporters (CCCs) such as Na+–Cl COTRANSPORTER (NCC), Na+–K+–2Cl COTRANSPORTER (NKCC),and K+–Cl COTRANSPORTER (KCC). Phosphorylated NCC and NKCC are activated. By contrast, phosphorylated KCC is inactivated. WNK4 also inhibits NCC surface expression by promoting its lysosomal routing and degradation. Moreover, WNK1 and WNK4 are known to exhibit inhibitory effects on RENAL OUTER MEDULLARY POTASSIUM CHANNEL 1 (ROMK1). Association of WNK1 with MAMMALIAN HOMOLOGUE of UNC-18c (Munc18c) at the WNK1 kinase domain in a phosphorylation-independent manner allows dissociation of Munc18c from syntaxin 4, leading to subsequent binding of syntaxin 4 with VESICLE-ASSOCIATED MEMBRANE PROTEIN 2 (VAMP2), vesicle fusion, and exocytosis. A similar effect can be achieved through phosphorylation of synaptotagmin 2 by WNK1. (b) The best understood member of the AtWNKs family is AtWNK8, which is involved in various processes, such as regulating intracellular signaling through G protein, floral transition, and proline accumulation in the face of osmotic stresses. AtWNK1 and AtWNK8 together coordinate the endocytosis of AtRGS1 and promote intracellular signaling. AtWNK8 and AtWNK9 possess contradictory roles in mediating plant response to environmental stress as they have opposing effects on proline accumulation. AtWNK2 and AtWNK5 crucially act as negative regulators of the flowering pathway and circadian clock via their inhibitory effects on EARLY FLOWERING 4 (ELF4), TIMING OF CAB EXPRESSION 1 (TOC1), and CONSTANS (CO). Meanwhile, AtWNK1 promotes CO and FLOWERING LOCUS T (FT) expression. (CO, FT, FLOWERING LOCUS C (FLC)), ELF4, TOC1, and VERNALIZATION INSENSITIVE 3 (VIN3) are genes participating in phytohormone signaling, photoperiod, and vernalization. They are important in different flowering pathways. The functions of AtWNK3, AtWNK4, AtWNK6, AtWNK7 and AtWNK11 are still unknown. Animal WNK kinases are depicted as blue ovals, and plant WNK kinases are depicted as green/orange ovals. Targets of the WNK kinases are other shapes, and open rectangles are cellular processes. Dotted lines represent possible physiological outcomes from protein interactions. Red lines represent inhibitory effects, while blue lines represent activation. Question marks depict unknown outcomes and functions of various proteins. EDM2, Enhanced Downy Mildew 2; APRR3, Arabidopsis Pseudo-Response Regulator 3; AtVHA-C, Vacuolar H+ ATPase Subunit C; UVRAG, UV Irradiation Resistance Associated Gene; PIK3C3, PI(3) Kinase Class III; PIK3R4, PI3K Regulatory Subunit 4; BECN1, Beclin 1; ULK1, Unc-51 Like Kinases 1.

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(a) WNK2 WNK3 WNK4 WNK1 MUNC18C

SYNTAXIN4

ROMK1 P P ULK1 SPAK OSR1 P SYNTAXIN4 P SYNAPTOTAGMIN 2 MUNC18C UVRAG

PPPPIK3C3 PIK3R4 Vesicle SYNTAXIN4 VAMP2 BECN1 fusion NCC NKCC KCC

or

NCC/NKCC lysosomal Exocytosis, Autophagy routing and degradation protein recycling

(b) AtWNK9 AtWNK8 AtWNK5 AtWNK2 AtWNK11

PP ? AtVHA-C EDM2

Proline ? accumulation ELF4 TOC1 CO FT FLC

Floral transition P AtRGS1 AtWNK1

P AtWNK7 AtWNK10 Endocytosis APRR3

Continues AtWNK3 AtWNK4 AtWNK6

Signaling Circadian ? rhythm triad (Lys-233) is located in strand b2 (subdomain I), instead of in with Glu-91 in helix C to interact with a- and b-phosphates of strand b3 (subdomain II) as in typical kinases such as Protein ATP. However, in mammalian WNK kinases, a cysteine residue Kinase A (PKA) (Lys-72) (Fig. 3c). It is this feature that (Cys-250) occupies the position of lysine in strand b3 (subdomain distinguishes WNK kinases from other protein kinases (Lys-233 II) at the position where Lys is expected for non-WNK kinases. To is indicated in Fig. 3c (right side) and as K233 black arrow in complicate matters further, in Arabidopsis, the mammalian- Fig. 2). In PKA, this catalytic lysine residue forms an ionic bond conserved Cys-250 (C250 arrow) is replaced by an asparagine

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(Fig. 2, black arrow C250). Mutation of Lys-233 fully abrogates kinase structure inferred here by modeling based on the animal WNK kinase activity of WNK kinases in rat (Xu et al., 2000). kinases (Figs 3, 4). For this review, structure prediction was Neither a crystal structure nor a solution structure for plant WNK performed using HHpred (S€oding et al., 2005), yielding a predicted kinases has been determined; therefore, we propose the plant WNK model of AtWNK6 based on the human WNK1 kinase template for

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Fig. 2 Molecular characterization of kinase domains of plant and animal WITH NO LYSINE (WNK) family members. From top to bottom: Arabidopsis thaliana WNK kinases: AtWNK1 (NP_187142.1), AtWNK2 (NP_188881.1), AtWNK3 (NP_680105.1); At(NP_200643.1); AtWNK5 (NP_566954.2), AtWNK6 (NP_001326380.1), AtWNK7 (Q8LST2.1), AtWNK8 (NP_568599.1), AtWNK9 (Q2V338.1), AtWNK10 (NP_176644.2); AtWNK11 (Q6ICW6.1); Physcomitrella patens WNK (PpWNK; A9TZB2); Marchantia polymorpha (MpWNK; A0A176W792); Homo sapiens WNK1 (HumanWNK1; Q9H4A3.2); Homo sapiens WNK2 (HumanWNK2; Q9Y3S1.4); Homo sapiens WNK3 (HumanWNK3; Q9BYP7.3); Homo sapiens WNK4 (HumanWNK4; Q96J92.1). The black arrows with amino acid symbol represent conserved residue position in rat WNK1. The red lines represent the active site motifs in the serine/threonine protein kinase domain: one is [LIVMFYC]-X-[HY]-X-D-[LIVMFY]-K-X(2)-N-[LIVMFYCT](3), whereby X is any amino acid residues and the other is a conserved ‘GTPEFMAPE’ motif (Taylor et al., 1995). The black lines represent the 12 consensus regions of the protein kinase domain. The multiple sequence alignment was generated using CLUSTAL W. Amino acids are background-shaded with their respective colors if their frequency in a column equals or exceedsa threshold of 75%. Generally, red is for acidic residues, blue for basic residues, and other colors are randomly assigned to residues to distinguish among them. The large black arrows denote amino acids discussed in the text (numbering based on human WNK1). Other residues indicated are elaborated in Fig. 3. thekinasedomainandonratWNK1fortheautoinhibitory(AI) WNK kinases. Similar to mammalian WNK kinases, plant WNK domain (Figs 3a, 4b). AtWNK6 kinase was the choice for compar- kinases possess a highly conserved AI domain that lies C-terminal to ison as it shares the highest sequence similarity to human WNK1 the kinase domain. This AI domain acts as an internal repressor of among all plant WNKs. However, it is important to note that the WNK kinase domain through an intramolecular interaction AtWNK11 kinase is characterized by an asparagine residue at (Xu et al., 2002; Wang et al., 2004). Kinase activity is suppressed position 161 (Fig. 3b), instead of a lysine (as Lys-158) that is essential until appropriate signals induce the release of the inhibitory for the binding with ATP (Fig. 3a) in the catalytic core. Whereas domain from its binding site. Hong-Hermesdorf et al. (2006) human WNK1 displays a unique topology of six-stranded b sheets successfully demonstrated the in vitro regulatory activity of the AI that position themselves into an almost complete b barrel, AtWNK6 domain in the Arabidopsis WNK kinase family: full-length seems to assume a common topology of five b-strands (Min et al., AtWNK8 and a kinase-dead version that possesses the putative 2004). Interactions within the catalytic core of predicted AtWNK6 AI domain display decreased auto-phosphorylation level and less resemble those of human WNK1: the four residues that coordinate transphosphorylation respectively. A solution structure of animal ATP binding in AtWNK6 are Lys-40, Asp-156, Lys-158 and Asn- WNK1 AI domain provides evidence that two a-helices orienting 161, corresponding to Lys-233, Asp-349, Lys-351 and Asn-354 against three b-strands (aA, aB, b1–3) exist among residues 480– respectively in human WNK1 (Fig. 3a,c). 572 (Fig. 4a,c, green box). Similarly, the predicted AtWNK6 AI domain shares 71 amino acids in a similar topology (Fig. 4b). The animal WNK1 AI domain binds Arg-Phe-Xaa-Val (RFxV)- 2. Autoinhibitory domain and auto-phosphorylation site containing peptides through the involvement of Asp-531, Ile-522, A highly conserved serine residue (Ser-382 in human WNK1, Phe-524, and the formation of a putative RFxV-binding groove Fig. 2) acts as an auto-phosphorylation site in the catalytic loop of between the b3–aA interface, which is coordinated by Phe-524, the kinase domain (Xu et al., 2002), across all animal and plant Asp-531, and Glu-539 (Moon et al., 2013) (Fig. 4a, RFxV-binding

(a) AtWNK6 (b) AtWNK11

Asp-156 Asp-159 Lys-40 Lys-43 Asn161 Lys-158 Mn Mn Asn-164 Asn-161

AMPPNP AMPPNP Fig. 3 Features of WITH NO LYSINE (WNK) Small lobe Large lobe kinase domains. (a, b) Predicted structure of the kinase domain of (a) AtWNK6 and (b) (c) Human WNK1 AtWNK11 obtained using the crystal structure (PDB: 5TF9) of human WNK1 in complex with Mn2+, AMPPNP and WNK476 as template (PDB: Asp-349 5TF9). The large and small lobes of the kinase domain are shown in yellow and marine colors. Catalytic core The catalytic core of WNK kinases possesses Lys-233 some atypical residues for the binding and catalysis of an adenine nucleotide. Those Lys-351 β3 Mn residues are shown in the sphere model. An β2 ATP analogue (AMPPNP) and a magnesium Asn-354 ion are shown with stick and sphere models. β1 Note that Arabidopsis WNK1–10 share catalytic core residues with human WNK kinases, while AtWNK11 lacks a lysine residue AMPPNP on the large lobe. (c) Crystal structure of the Small lobe Large lobe kinase domain of human WNK1 (PDB: 5TF9).

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(a) (b)

β1 β1 β3 β2 β3 F524 α β2 αB B F393

αA F526 αA E408 F395

D531 D400 E539 Putative RFxV binding groove

(c) P-rich S-rich P-rich Autoinhibitory Kinase domain Coil Coil Human WNK1 domain 2382 218–494 480–572 ?

Autoinhibitory Kinase domain Coil AtWNK6 domain 567 28–285 363–434 522–554

AtWNK11 Kinase domain 314 20–275 Fig. 4 Comparison between the autoinhibitory domains of animal and plant WITH NO LYSINES (WNKs) and comparison of domain architecture among animal and plant WNKs. (a) Autoinhibitory domainof rat WNK1 as derived from solution NMR. The autoinhibitory domain of rat WNK1 comprises two linking a-helices (aA and aB) that lie adjacent to three b strands (b1, b2 and b3). A linker connects between b1 and b2, as well as between b2 and b3 (Moon et al., 2013). Residues depicted in red, purple, or green color represent conserved amino acids that potentially form RFxV binding groove. (b) Predicted structure of the autoinhibitory domain of AtWNK6 obtained from HHpred, using rat solution structure of the WNK1 Autoinhibitory Domain (PDB: 2LRU) as a template. (c) Illustration comparing the domain structure of WNK1, AtWNK6, and AtWNK11. Represented are the kinase domain, the autoinhibitory domain, predicted coiled-coil domains, and proline (P)- and serine (S)-rich regions. The question mark represents the unknown number of residues for the coiled-coil region in human WNK1. The red circle illustrates a putative RFxV binding groove.

cluster is within the red circle). In human WNK1 kinase, there are other is at the extreme C terminus (Fig. 4c). WNK1 kinase is a five RFXV motifs (one in the kinase domain and four in the C tetramer, implying the role of coiled-coil domains in oligomeriza- terminus) (Min et al., 2004). Intriguingly, Arabidopsis WNK tion. Moreover, coiled-coil domains participate in vesicle tethering kinases do not possess any RFxV motifs. However, Arabidopsis (Cheung & Pfeffer, 2016). For example, and pertinent to our WNK kinases still maintain the same conserved residues as animal speculation on a role in protein trafficking below, the local WNKs; namely, Phe-393, Phe-395, Asp-400 and Glu-408 in unwinding of coiled-coil domains in Golgin GCC185 enables it to AtWNK6 (Fig. 4b). This begs the question of what mechanism and contact vesicles and guide them toward the Golgi membrane (Yu binding site the Arabidopsis WNK kinases utilize to perform auto- et al., 2010). Only Arabidopsis WNK3, -6, -7, -8, -10, and Oryza inhibition. Besides the auto-inhibition, RFxV motifs act as sativa WNK2 and -4 show a predicted coiled-coil domain at the recognition sites for a conserved carboxy-terminal domain present extreme C terminus with low similarity compared with that of in OSR1 and SPAK (Villa et al., 2007). Moreover, plants lack mammalian WNK kinases (Supporting Information Fig. S1). SPAK/OSR1 homologues. Therefore, it calls into question Moreover, the short coiled-coil domain that is most adjacent to the whether plant WNK kinases interact with a completely different AI domain is not present in any of the plant WNK kinases (Hong- set of downstream targets compared with animal WNK kinases. Hermesdorf et al., 2006). Generally, C-terminal domains of plant WNK kinases are widely disparate and may confer structural and functional differences between them. 3. Coiled-coil domains and myristoylation There are several N-myristoylation sites toward the N terminus Mammalian WNK kinases contain coiled-coil domains of AtWNK8, indicating the possibility of post-translational (McCormick & Ellison, 2011). This a-helical coiled-coil (Fig. 4c, modification and promotion of reversible membrane binding. orange boxes) is best characterized by a heptad repeat pattern of Moreover, recently, a study using tandem mass spectrometry hydrophilic and hydrophobic residues that together construct the identified that human WNK1 undergoes b-O-linked N- oligomer interface (Burkhard et al., 2001). Sequence analysis of acetylglucosamine modification (Nagel et al., 2013). In fact, many human WNK1 reveals two coiled-coil domains in the C-terminal protein kinases, phosphatases and Ga proteins are known to possess extension: one is located C-terminal to the AI domain, and the N-terminal myristoylation sites (Braam & Verhaar, 2007).

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There is essentially only one published account of plant WNK 4. Special member WNK11 kinase gene expression (Wang et al., 2008). Arabidopsis WNK There is a concern in the community that AtWNK11 is a kinase gene expression levels in various tissues differ widely in pseudogene. We debunk this idea because WNK11 is not only mature plants. AtWNK3, AtWNK4 and AtWNK5 shared a similar present inArabidopsisbut alsoin soybean,rice, and otherplants.The pattern in root, stem, and leaf; however, AtWNK4 and AtWNK5 PANTHER Classification System identifies uncharacterized soy- levels are considerably higher than that of AtWNK3. AtWNK1 was bean proteins (XP_003553016 and XP_003530782) under the more highly expressed in stem than in root. AtWNK2 was mildly subfamily of WNK11-related proteins and as AtWNK11’s least- expressed in flower, and AtWNK9 was exclusively expressed in root. diverged orthologues. They all share one common ancestor based on Meanwhile, AtWNK7 level of expression was barely detectable the PANTHER phylogenetic analysis (Mi et al., 2013). A mesoscale across root, stem, leaf, and flower. AtWNK8 was predominantly abscisic acid (ABA) interactome demonstrated that AtWNK11 expressed in stem and faintly in leaf, while AtWNK10 was mostly interacts with AtWNK2 (Table S1) (Lumba et al., 2014), indicating expressed in leaf and only slightly in root. Nevertheless, such an that both of them may be involved in a kinase cascade and one WNK expression pattern is inconsistent between Arabidopsis and rice. kinase could control the activity of the other. Moreover, AtWNK11 OsWNK5 and OsWNK6 were most prominently expressed in roots does not possess the auto-inhibitory domain. Only the kinase at levels 10-fold those of other tissues. In addition, other OsWNKs domain is observed within AtWNK11, from residue 20 to residue that largely expressed in roots include OsWNK2 and OsWNK9. 275 (Fig. 4c). The uniqueness in sequence is enough to propose However, OsWNK9 shows mild transcription levels in other organs confidently that its mode of intramolecular regulation is different as well, unlike in Arabidopsis. OsWNK3, OsWNK4 and OsWNK7 from other plant WNK kinases. generally maintained low expression levels across culm, panicle, root, and leaf. OsWNK8 exhibited considerable transcript level in leaf. OsWNK1 was more highly expressed in leaf and root (Manuka III. Phylogeny–evolutionary relationships et al., 2015). WNK-family kinases exist in the Eukaryote subgroups Opisthokonts Because little has been published on WNK kinase distribution in (animals and fungi), Amoebozoa, Archaeplastida (plants) and plant organs and within plant cells, we augment the published Excavata (unicellular protists), but not in Prokaryote and Archaea literature with our own analyses here (Fig. S2). The pattern of kingdoms. This gene conservation pattern suggests that WNK-family expression shown in Fig. S2(a) incorporates annotation from the kinases emerged in a eukaryotic ancestor, before the separation of the Plant Ontology Consortium (http://www.plantontology.org/) major eukaryotic groups. Most WNK kinases in Eukaryotes conserve indicating the various tissues (Zimmermann et al., 2004). There the unconventional catalytic core residues, the auto-phosphorylation are six main groups in Fig. S2(a): callus, cell culture/primary cell, site, and the AI domain, suggesting that the catalytic action of WNKs seedling, inflorescence, shoots, and roots, followed by the corre- and the regulation by auto-phosphorylation are fundamentally sponding subgroups. A comparison based on absolute expression conserved across eukaryotic lineages. WNK kinases in plants and values is not feasible; only expression patterns among individual some other lineages lack the carboxyl-terminal RFxV motif that AtWNK kinases are comparable. For example, WNK6 is expressed forms the intermolecular interaction with the AI domain, and the in inflorescence more than in photosynthetically inactive tissues regulatory mechanism of those WNKs is yet to be elucidated. The (Fig. S2a, AtWNK6 column). Among this kinase family, AtWNK9 diversification of plant WNK kinases is shown in Fig. 5. Primitive absolute expression is highest (Fig. S2a, WNK9 column). Xie et al. plants, Physcomitrella patens (moss) and Marchantia polymorpha (2014) found that overexpression of AtWNK9 in the presence of (liverwort), have single WNK genes. The prototypical WNK kinase ABA inhibits primary root elongation. The abundance of AtWNK9 gene repeatedly duplicated and diversified into four subgroups in the in roots, and no other tissues, suggests that future analysis should be short period of seed plant evolution. Arabidopsis and rice genomes focused on its importance in root biology. Microarray heat-map encode, respectively, 11 and 9 WNK kinase genes that fall in four matrix reflects gene expression levels upon exposure to different subgroups, implying their class-specific functions are well constrained conditions or under temporal evolution (Fig. S2b). For instance, a in spermatophyte species. Genetics studies in Arabidopsis partially potential role of AtWNK7 in nitrate regulation is demonstrated by support the idea of distinct functions between WNK subgroups in experiments with potassium nitrate or studies with limiting development and stress responses (see Section V). ammonium nitrate. Future research could make use of such data for hypothesizing the physiological importance of WNK kinases in plant response to various environmental treatments. IV. Plant WNK kinase distribution and regulation of WNK expression and activity V. Functions of WNK family members in physiology Soybean WNK1 (GmWNK1) expression is specifically associated and development with root cells involved in lateral root formation. As shown in Fig. 1(b) and Table S1, Arabidopsis WNK8 interacts with 1. Regulation of ion transport in plants Enhanced Downy Mildew 2 (EDM2) in the nucleus, which subsequently influences the expression of the floral repressor AtWNK8 phosphorylates subunit C of the Vacuolar H+-ATPase gene Floral Locus C (FLC), At5g10140) (Tsuchiya & Eulgem, (V-ATPase, AtVHA-C) in Arabidopsis at amino acid residues Ser- 2010). 116, Thr-204, Ser-211, Ser-212, Ser-253 and Ser-331 (Hong-

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E1ZGB9 CHLV A Algae

WNK5 ORYSJ

96 Group 4

WNK11 ARATH 99 100

WNK2 ARATH

WNK9 ARATH 95 WNK1 ARATH WNK1 ORYSJ

WNK3 ORYSJ

86 WNK8 ORYSJ WNK7 ORYSJ Group 3 WNK5 ARATH

97

80

WNK4 ARATH

WNK2 ORYSJ WNK3 ARATH A0A176W792 MARPO A9TZB2 PHYPA D8SU99 SELML

D8SWL2 SELML Group 2 D8T849 SELML D8T8A 1 SELML WNK7 ARATH WNK6 ARATH

67 WNK9 ORYSJ WNK6 ORYSJ WNK4 ORYSJ Group 1

WNK10 ARATH WNK8 ARATH

Fig. 5 A phylogeny of plant WITH NO LYSINE (WNK) proteins. This maximum-likelihood tree was constructed using the kinase domain sequences of selected plant and algal WNKs. WNKs from representative species are shown in the tree with gene names (Arabidopsis and rice) or UNIPROT accession numbers (other species). The number at each node represents the percentage of bootstrap supports in 100 replicates. Species names in the tree: ARATH, Arabidopsis thaliana; ORYSJ, Oryza sativa Japonica; SELML, Selaginella moellendorffii; PHYPA, Physcomitrella patens; MARPO, Marchantia polymorpha; CHLVA, Chlorella variabilis. The tree was rooted with algal WNKs sequences.

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Hermesdorf et al., 2006). The interaction appears specific, in that suppressing the plant’s oxygen-scavenging system (Zhang et al., no other WNK family member, including AtWNK10, which bears 2013). the highest similarity to AtWNK8, is able to interact with AtVHA- Overexpression of soybean GmWNK1 in Arabidopsis plants C in the yeast two-hybrid system. Moreover, the homologues of enhances resistance toward exogenous ABA as well as lowers plant subunit C from various other organisms such as Manduca sexta, sensitivity to ionic stresses and osmotic stresses induced by Caenorhabditis elegans and Saccharomyces cerevisiae are phosphory- mannitol (Wang et al., 2011). Moreover, the level of endogenous lated by AtWNK8, portending a conserved function across species. ABA increases upon overexpressing GmWNK1 in Arabidopsis and Phosphorylation of V-ATPase subunit C is proposed to be crucial soybean. GmWNK1 interacts with GmCYP707A1 – a putative for the fully assembled complexes of V1 and V0 sub-complexes ABA 80-hydrolase from soybean (Wang et al., 2010), essential for (Curtis et al., 2002). Hence, AtWNK8 might act in protein-kinase- ABA catabolism (Finkelstein, 2013). mediated signaling of V-ATPases to control V-ATPase assembly, and thus regulate intracellular pH homeostasis. 3. WNK family members are involved in circadian rhythm and flowering time 2. Hormonal regulation to osmotic and ionic stresses Arabidopsis WNK family modulate the circadian clock High soil salinity with concomitant reduction in soil water (Nakamichi et al., 2002). To satisfy the criteria for being controlled potential is one of the most restricting abiotic stresses of plants by a circadian clock, an observed rhythm must be synchronized by (Pardo, 2010). High salinity generates two types of stressors in environmental signals like day and night shifts (Dunlap, 1999). As plants. First, an ensuing water deficit acts as a signal of osmotic such, the level of RNA transcripts for AtWNK1, AtWNK2, stresses (Munns & Tester, 2008). This osmotic stress stimulates AtWNK4, and AtWNK6 are influenced by circadian rhythms the production and accumulation of ABA – a plant stress- (Fig. 1b). AtWNK1 phosphorylates the putative clock component responsive hormone – and results in a lower stomatal conduc- Arabidopsis Pseudo-Response Regulators 3 (APRR3) – one of the tion, and thus limits water loss (Roy et al., 2014). Second, an ion members of the APRR1/TOC1 quintet that is implicated in imbalance forms due to toxic concentrations of sodium (Na+)in coordinating higher plants’ circadian rhythms (Murakami-Kojima the cytoplasm and a concurrent shortage of potassium (K+) et al., 2002). Both AtWNK1 and APRR3 display coincidental (Greenway & Munns, 1980), leading to metabolic damage in rhythmic transcriptome expression. The OsWNK1 transcript root and leaf tissues (Lynch et al., 1995), and consequently level has a cycling pattern similar to that of OsPRR1 – a well- necrosis (Roy et al., 2014). Interestingly, it is observed that two established marker gene for circadian regulation (Kumar et al., members of the AtWNK kinase family –AtWNK8 and 2011) – suggesting mediation of flowering time (Wang et al., AtWNK9 – respond to ABA in an almost opposite manner. 2008). The effect of AtWNK1 on flowering time contrasts with that AtWNK8 negatively influences the plant’s ability to cope with of AtWNK2/5/8: AtWNK1 promotes CONSTANS and both osmotic stress and ion imbalance in vivo. Overexpression of FLOWERING LOCUS T expression while AtWNK2/5/8 suppress AtWNK9 promotes plant survival under drought stress compared them. Based on the interaction of WNK1 and WNK4 in animals, with wild-type. A wnk8 null mutant resistant to sorbitol and AtWNK2/5/8 might be negatively regulated by AtWNK1 (Wang sodium chloride (NaCl) treatments (Zhang et al., 2013). A et al., 2008). Tsuchiya & Eulgem (2010) showed that Arabidopsis proposed explanation for this phenomenon lies in the concurrent EDM2 (a protein known to participate in plant disease resistance) is proline accumulation under both salt and osmotic stress. Peng phosphorylated by AtWNK8 in the nucleus before it regulates the et al. (2012) revealed that exogenous ABA treatment causes expression of the floral repressor gene FLC, overall promoting floral proline accumulation in Arabidopsis. Stronger resistance to transition. The detailed mechanisms on WNK kinase roles in osmotic pressure in wnk8 may depend on a significantly higher flowering and circadian rhythm remain unknown. proline concentration produced, while tolerance toward ionic stress seems to be independent of proline content (Zhang et al., 4. WNK kinases in protein trafficking 2013). By contrast, wnk9 has the lowest proline accumulation compared with that of wild-type (Xie et al., 2014). Therefore, it Our view is that WNK kinases have two functions in signal is possible that AtWNK8 and AtWNK9 respond to ABA and transduction through protein trafficking: (1) to communicate the subsequently play contrasting roles in regulating the synthesis of intensity of extracellular signaling, and (2) to dictate the physio- proline in plant tissues. The relationship between AtWNK9 and logical outcome under the influence of a signal. As for function 1, ABA was demonstrated by Xie et al. (2014): both negative AtWNK kinases transduce both the dose and duration of signals, regulators (ABI1 and ERA1) and positive regulators (ABF3 and and this is best elaborated using plant WNK kinases (Urano et al., ABI3) of ABA signaling have expression levels that are dependent 2012, 2013; Fu et al., 2014). As for function 2, the trafficking route on the amount of AtWNK9 in the cell. The relationship between itself is influenced by WNK kinases, and this is best elaborated AtWNK8 and ABA, however, is not as clear. The Arabidopsis using examples on animal WNK kinases. wnk8 mutant exhibits a higher activity of enzymes involved in The role in protein trafficking is better understood for animal scavenging reactive oxygen species (ROS), such as catalase and WNK kinases than in plants; as such, it is important that we delve peroxidase, making wnk8 more effective in tolerating environ- into the details for WNK kinases in trafficking in animals so that mental stresses. As a result, AtWNK8 may be involved in plant WNK researchers may extrapolate. WNK kinase involvement

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in animal sorting mechanisms includes both phosphorylation- In Arabidopsis, WNK kinases function in membrane trafficking dependent and -independent mechanisms. For example, WNK1 (Urano et al., 2012). AtWNK8 phosphorylates AtRGS1 at its C- was found to participate in the negative regulation of autophagic terminal, both necessary and sufficient for endocytosis. AGB1/ trafficking (Kankanamalage et al., 2017). Human WNK4 inhibits AGG1 first recruits AtWNK8 before AtRGS1 endocytosis occurs. NCC activity through minimizing NCC on the plasma membrane Several questions remain unanswered: (1) What are the events that (Cai et al., 2006; Golbang et al., 2006) by redirecting it from the follow the phosphorylation of AtRGS1 by AtWNK8? The possible plasma membrane to a lysosomal compartment for degradation and fates are degradation, recycling back to the plasma membrane, or overall preventing the insertion of NCC into the plasma membrane forming cytoplasmic signalosomes. (2) What WNK kinase-based (Fig. 1a). WNK4 expression strengthens the association between endocytic mechanisms internalize AtRGS1? Investigations con- NCC and the AP3 complex to enhance cargo transport to lysosomes ducted on animal WNKs in recent years highlight their roles in at (Subramanyaet al.,2009).Zhouet al.(2010)proposedthatWNK4 least two of the various endocytic mechanisms: clathrin-mediated phosphorylates the N terminus of NCC in the trans-Golgi network endocytosis and caveolae-mediated endocytosis. (3) How is to modify its interaction with sortilin, a lysosomal targeting AtWNK8 recruited by the Gbc dimer? It is reasonable to propose receptor.Thethree proteinsformacomplexthat potentiallyrecruits that AtWNK8 undergoes autoinhibition by intrinsic domains clathrin and AP1 or AP3 for the sortilin-mediated lysosomal before it comes into contact with AtRGS1, phospholipids, and the pathway. WNK1 functions similarly to WNK4, as it induces the Gbc subunit and then undergoes a conformational change that self- endocytosis of the distinct ATP-regulated K+ channel ROMK1. activates. (4) How does WNK kinase-based trafficking of the G Interestingly, this process requires a clathrin adaptor molecule, protein components control the dynamics of cellular outcomes. autosomal recessive hypercholesterolemia (ARH), and possibly Studies on G-protein signaling in Arabidopsis highlight an entails WNK1 phosphorylating ARH (Fang et al., 2009). WNK1 emergent property called dose–duration reciprocity (Fu et al., directly interacts with the priming protein Munc18c, which 2014). A low dose of signal, such as a hormone, administered over a facilitates insulin granule fusion (Fig. 1a) (Oh et al., 2005, 2007). long duration elicited an equivalent response as a high dose However, Munc18c does not serve as a substrate for WNK1 in administered in a short burst. AtWNKs intriguingly partake in the kinase activity assay because the kinase-dead WNK1 mutant retains tight control of this process by regulating the proportion of the binding capacity to Munc18c, implying other functions of endocytosed AtRGS1, and thus the extent of signal transduced. WNK1besidesphosphorylationinsolubleNSFattachmentprotein AtRGS1–WNKs interaction modify in accordance with changes in receptor(SNARE)complexassembly.Munc18cbindsSyntaxin 4,a glucose concentrations and time scales (Fu et al., 2014). Specifi- SNARE component, to reverse the inhibitory state of Syntaxin 4 cally, high concentrations of D-glucose (for instance, 6% for 0.5 h) (Fig. 1a). Subsequent dissociation of Munc18c–Syntaxin 4 com- swiftly signal through stimulation of AtRGS1 phosphorylation by plex is an essential step for vesicle fusion to occur. Stimulus-induced AtWNK8. Meanwhile, low and prolonged sugar concentration (for tyrosine phosphorylation of Munc18c dissociates it from Syn- instance, 2% for 5 h) gradually activates Arabidopsis glucose taxin 4, allowing Syntaxin 4 to associate with vesicle-associated response machinery by promoting AtWNK1 phosphorylation of membrane protein 2 (VAMP2). WNK1–Munc18c complexes are AtRGS1. The overall effect enables the cells to produce comparable crucial for VAMP2 association with Syntaxin 4, and WNK1 may response in face of both fleeting, high-intensity signals and act as a scaffolding protein to recruit Munc18c for other kinases or extended, low-intensity signals. The proportion of internalized phosphatases (Fig. 1a) (Oh et al., 2007). Intriguingly, Munc18c is a AtRGS1 is sensitive to the varying AtRGS1 to AtWNK kinase member of the Sec1–Munc18 (SM) family of proteins that widely ratios in a nonlinear manner: as the system reaches saturation, a spans plants, yeasts, and mammals. In plants, members of the SM lower ratio of internalized AtRGS1 is observed than when there are family, such as SEC11, interact with syntaxins; for example, Qa- a lower number of AtRGS1 molecules. Less substrate projects a SNARE SYP121 (Syntaxin of Plants 121) (Karnik et al., 2013), high proportion of phosphorylated AtRGS1, while a high number which participates in cellular responses to the stress hormone ABA of AtRGS1 projects a low proportion of phosphorylated AtRGS1 (Eisenach et al., 2012), as well as binds to K+ channels to modulate (Liao et al., 2017a,b). From a physiological perspective, this solute uptake for plant growth (Honsbein et al., 2009). In complex dynamics of AtRGS1 pool sizes imparts upon the plant the Arabidopsis thaliana, there are six SM members: VPS33, VPS45, ability to distinguish shadows from flickers and from the end of day SEC11,SEC12,SEC13,andaprotein thatdemonstrateshomology (Liao et al., 2017a) and for cells distal to the photosynthetic centers, to the yeast SM protein Sly1p (Sutter et al., 2006). AtVPS45 such as roots, to receive information of the state of autotrophy positively regulates the SYP41/SYP61/VTI12 trans-Golgi network (Tunc-Ozdemir et al., 2018). SNARE complex (Zouhar et al., 2009). Meanwhile, SEC11 A possible downstream effector of Arabidopsis G-protein interacts with Qa-SNARE SYP111 to induce vesicular fusion signaling, as facilitated by AtWNKs, involves Arabidopsis Hexok- during cytokinesis (Waizenegger et al., 2000). Therefore, further inase 1 (AtHXK1) (Moore et al., 2003; Chen & Jones, 2004; investigations should beconductedto explorewhether plant WNKs Urano et al., 2012). AtRGS1 and AtHXK1 communicate through interact with any member of the SM family to regulate vesicular cooperative regulatory loops to arrive at a conclusive response to transport and secretion, especially in the context of salt and drought glucose. Such a process occurs downstream of AtRGS1 endocytosis stress or under the influence of ABA. Moreover, the nature of such facilitated by AtWNK8. Moreover, AtRGS1 and AtHXK1 interactions, be it kinase dependent or kinase independent, should Interacting Protein 1 acts as a physical scaffold for these two be evaluated. sensors.

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Recently, transcriptomic analyses using Affymetrix microarrays 5. WNK kinases and ion channel regulation and Gene Ontology to monitor rice root responses to short- and Given the parallels of regulatory components and downstream long-term potassium deficiency revealed that 4.2% of the 2896 effector proteins, a common theme of regulation and signal differentially expressed genes possess kinase activity with kinases transduction between plants and animals is tempting. Hisamoto from the Ca2+/calmodulin-dependent protein kinase class and STE et al. (2008) discovered the role of WNK in phosphorylating the group displaying greater amount of transcriptional changes SPAK/OSR1 orthologue, germinal center kinase 3 (GCK-3) in compared with other groups (Ma et al., 2012). In particular, C. elegans. wnk-1 or gck-3 mutant worms have an undeveloped OsWNK5, OsWNK7, and OsWNK8 transcripts are upregulated, excretory canal – a component of the nematode renal system – due while OsWNK9 is downregulated. It is suggested that these kinase to failure to detect changes in extracellular osmolarity (Hisamoto genes are transcriptionally regulated by potassium deficiency, et al., 2008; Kupinski et al., 2010; Kolotuev et al., 2013). As a indicating their potential involvement in regulating K+ uptake and result, mutated GCK-3 proteins are unable to phosphorylate and K+ homeostasis during periods of deprivation. Interestingly, negatively regulate CLH-3b, which is a chloride channel OsWNK3,OsWNK6, and OsWNK7 transcripts increase under salt identified in worm oocytes (Denton et al., 2005; Hisamoto stress (Manuka et al., 2015). It is noteworthy that salt stress caused et al., 2008) and a member of the CLC-1/2/Ka/Kb chloride by excessive accumulation of Na+ at the root surface inhibits K+ channel family. Interestingly, the CLC chloride channel family uptake by the root and inevitably causes K+ deficiency (Zhu, 2001). also exists in plants. There are seven Arabidopsis AtCLC ion For that reason, sodium-tolerant plants must preserve K+ nutrition channels (Hechenberger et al., 1996) localized to the tonoplast (Liu & Zhu, 1998) by turning on high-affinity potassium transport (De Angeli et al., 2006), to chloroplast membranes, and to Golgi and switching off low-affinity potassium transport (Ashley et al., vesicles (von der Fecht-Bartenbach et al., 2007; Marmagne et al., 2005). Furthermore, K+ deficiency leads to acute rise in photo- 2007). A member of the Arabidopsis CLC family, AtCLCc, located synthetically generated electrons, prompting increased ROS on the tonoplast, is involved in stomatal aperture (Jossier et al., production (Cakmak, 2005). AtWNK9 abundance correlates with 2010). AtCLCc is in guard cells, and mutant AtCLCc plants do not the abundance of ROS-scavenging gene transcripts (Xie et al., respond to light-induced stomatal opening and ABA-induced 2014). Taken together with the fact that K+ deficiency induces ABA stomatal closure (Jossier et al., 2010). When the stomata opens, accumulation, we propose that WNK family members in plants guard cells import more Cl into their vacuoles to balance K+ enhance the plant oxidative defense system and the expression of intake (Lasceve et al., 1987). However, with impaired Cl potassium transporters in response to the plant hormone ABA, transport in AtCLCc mutants, Cl is not shuttled into the vacuole, which is synthesized in response to K+ deficiency. Hence, future while simultaneously Cl does not move from the vacuole to cell research should identify potassium transporters that are regulated cytoplasm to enable vacuole depolarization and thus stomatal by plant WNKs. Potential candidates for K+ transporters in plants closure. Other members of the Arabidopsis CLC family, such as include high-affinity potassium transporters, which function in AtCLCs, AtCLCd, and AtCLCg, preferentially transport Cl potassium-poor environments, such as the KT/KUP/HAK family compared with AtCLCa, which prefers nitrate (Wege et al., 2010; members (Grabov, 2007). Twenty-seven KT/KUP/HAK genes Zifarelli & Pusch, 2010). Therefore, these channels participate in exist in rice and 13 in Arabidopsis (Grabov, 2007; Gupta et al., physiological processes that need Cl transport, such as stomatal 2008). Expression of the Arabidopsis HAK5 (AtHAK5) in the movement and salt tolerance by regulating Cl homeostasis epidermis of roots is activated by low extracellular K+ concentration (Teakle & Tyerman, 2010). Furthermore, tolerance toward salt (Ahn et al., 2004; Gierth, 2005). Inhibition of ROS synthesis using stress necessitates compartmentalization of Na+ and Cl in plant an NADPH oxidase inhibitor prevents the transcription of HAK5 vacuoles to prevent toxic accumulation of ions in the cytoplasm. (Shin & Schachtman, 2004), suggesting K+ stress involves ROS Rice and soybean CLC family members (OsCLC-1 and GmCLC- signaling cascades (Amtmann et al., 2005). As a result, WNK 1) were stimulated under NaCl treatment to improve tolerance to family kinases may also control the surface expression of potassium NaCl by sequestering Cl in the vacuole (Li et al., 2006; transporters, eventually influencing downstream events such as Nakamura et al., 2006). ROS production and intracellular K+ homeostasis in response to While plants lack homologues of SPAK/OSR1, there still is a environmental K+ stress via intertwining networks. Future research plant analogy based on AtWNK kinases and downstream Cl employing a trafficking inhibitor like Brefeldin A (Subramanya channels, G proteins, and drought stress. gpa1 mutants are et al., 2009) or dominant-negative dynamin (Cai et al., 2006; hypersensitive to ABA inhibitory effects on seed germination, Golbang et al., 2006) may elucidate the relationship between plant early seedling development and root elongation, and ABA- WNKs and ion transporters. induced gene expression. By contrast, these mutants are hyposensitive to ABA inhibition of stomatal opening and inward VI. Say no more? Still many questions to be answered K+ channels (Wang et al., 2001). wnk9 mutants are hyposensitive to ABA inhibition of stomatal opening and root elongation. Plant WNKs present a diverse repertoire of regulatory kinases that Further studies should examine if AtWNKs respond to ABA modulate various plant physiological processes. Nevertheless, under drought stress by regulating signal transduction via several important questions remain unanswered. The functional AtRGS1–AtGPA1 and thus modifying the expression of plant divergence and redundancy between WNK kinase subgroups is AtCLC family members. completely unknown. The complete repertoire of substrate and

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