The soybean rootspecific protein kinase ... - Wiley Online Library

The Plant Journal (2010) 64, 230–242

doi: 10.1111/j.1365-313X.2010.04320.x

The soybean root-specific protein kinase GmWNK1 regulates stress-responsive ABA signaling on the root system architecture Yingxiang Wang1,2,*, Haicui Suo1, Yan Zheng1, Kaidong Liu1, Chuxiong Zhuang1, Kristopher T. Kahle3, Hong Ma2,4 and Xiaolong Yan1 1 Root Biology Center, South China Agricultural University, Guangzhou 510642, China, 2 Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China, 3 Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA, and 4 Department of Biology and the Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA Received 6 April 2010; revised 29 June 2010; accepted 28 July 2010; published online 1 September 2010. * For correspondence (fax +86 21 55664187; e-mail [email protected]).

SUMMARY In humans, members of the WNK protein kinase family are osmosensitive regulators of cell volume homeostasis and epithelial ion transport, and mutation of these proteins causes a rare inherited form of hypertension due to increased renal NaCl re-absorption. A related class of kinases was recently discovered in plants, but their functions are largely unknown. We have identified a root-specific WNK kinase homolog, GmWNK1, in soybean (Glycine max). GmWNK1 expression was detected in the root, specifically in root cells associated with lateral root formation, and was down-regulated by abscisic acid (ABA), as well as by mannitol, sucrose, polyethylene glycol and NaCl. In vitro and in vivo experiments showed that GmWNK1 interacts with another soybean protein, GmCYP707A1, which is a key ABA 8¢-hydroxylase that functions in ABA catabolism. Furthermore, 35S-GmWNK1 transgenic soybean plants had reduced lateral root number and length compared with wild-type, suggesting a role of GmWNK1 in the regulation of root system architecture. We propose that GmWNK1 functions to fine-tune ABA-dependent ABA homeostasis, thereby mediating the regulation of the root system architecture by ABA and osmotic signals. The study has revealed a new function of a plant WNK1 gene from the important staple crop soybean, and has identified a new component of a regulatory pathway that is involved not only in ABA signaling, but also in the repression of lateral root formation by an ABA-dependent mechanism distinct from known ABA signaling pathways. Keywords: GmWNK1, ABA, root system architecture, GmCYP707A, soybean, osmotic stress.

INTRODUCTION The root system architecture (RSA), the spatial configuration of the root system in the soil, is profoundly affected by water and nutrient availability, and also appears to be critically important for edaphic stress in many crop species, particularly legumes such as soybean and common bean (Liao et al., 2001; Zhao et al., 2004; Malamy, 2005; De Dorlodot et al., 2007). Genetic studies have indicated that the RSA is closely associated with several major quantitative trait loci (QTL) that can be used to facilitate selection and breeding for optimal RSA (see review in De Dorlodot et al., 2007). A promising example of so-called ‘root breeding’ is the 230

development of a number soybean breeding lines with superior root characteristics that enable improved adaptation to acid soils (Yan et al., 2006). Of the factors that control the total RSA, the postembryonic initiation and growth of lateral roots are among the most important steps that allow the plant to incorporate information from the environment to mediate adaptation of the RSA to soils. The RSA (or lateral formation) is also greatly influenced by complex interactions among hormones, root development and the environment (Forde and Lorenzo, 2001; Casimiro et al., 2003; Lopez-Bucio et al., 2003; ª 2010 The Authors Journal compilation ª 2010 Blackwell Publishing Ltd

GmWNK1 regulates root system architecture in soybean 231 Malamy et al., 2005; De Dorlodot et al., 2007). Abscisic acid (ABA), the universal stress-responsive hormone of higher plants, has been implicated in the regulation of lateral root development at specific stages in response to environmental stresses (De Smet et al., 2006). For example, a number of studies have shown that ABA plays an important role in repressing lateral root development (De Smet et al., 2006), as ABA-deficient plants produce larger root systems than isogenic wild-type plants (Deak and Malamy, 2005). The inhibition of lateral root development by ABA could not be rescued by exogenous auxin (De Smet et al., 2003), which promotes the formation of lateral roots. On the other hand, auxin was able to induce lateral root initiation in an ABAinsensitive mutant, abi3 (Brady et al., 2003). Furthermore, ABA also plays an important role in mediating the effects of nutrients, such as NO3 , on lateral root formation (Signora et al., 2001). Recent studies have shown that pathways promoting lateral root dormancy are controlled by genes that are distinct from those involved in seed dormancy (Zhang et al., 2007). More interestingly, mutants of the FCA gene, a key regulator of flowering in Arabidopsis, showed reduced sensitivity to the inhibitory effect of ABA on lateral roots, supporting a role for FCA in ABA signaling for lateral root development (Macknight et al., 1997; Simpson et al., 2003). Mild osmotic stress conditions also inhibited lateral root formation in an ABA-dependent way (Deak and Malamy, 2005; Malamy, 2005; Xiong et al., 2006; Qi et al., 2007). This inhibition does not occur in the lateral root development2 (lrd2) mutant or two ABA-deficient mutants (Deak and Malamy, 2005; Malamy, 2005). Recent molecular cloning has shown that LRD2 is the same as LACS2, which encodes a long-chain acyl CoA synthetase and is essential for leaf cutin formation. Moreover, a recent study provided new insights into the regulation of Arabidopsis lateral root inhibition by an ABA-dependent signal from the shoot to the roots (MacGregor et al., 2008). However, our knowledge on the mechanistic basis of regulation of the root system architecture in response to environmental changes associated with ABA signaling remains rudimentary. The WNK kinases are a family of serine/threonine kinases with a unique substitution of cysteine for lysine in b-strand 3 of kinase subdomain II (Xu et al., 2000) (WNK indicates ‘with no lysine’). In many other members of the protein kinase superfamily, this lysine is important for ATP-binding and the catalysis of phosphoryl transfer. In WNK kinases, the lysine that fulfills this essential function is situated in the phosphate anchor ribbon of kinase subdomain I. Mutations in WNK genes in humans cause a Mendelian-inherited form of NaCl-sensitive hypertension (Wilson et al., 2001). Many members of the WNK family are NaCl-sensitive kinases that regulate epithelial ion transport and cell volume homeostasis in response to osmotic stress via their action on multiple ion transporters and channels (Gagnon et al., 2006; Kahle

et al., 2006). Four WNK kinases exist in humans, and single WNK homologs exist in Drosophila melanogaster and Caenorhabditis elegans. Ten WNK-related genes in Arabidopsis and seven WNK-related genes in rice have been identified, implying a prominent role for this kinase family in plants (Nakamichi et al., 2002; Wang et al., 2008). In Arabidopsis, AtWNK1 interacts with and phosphorylates the clockassociated protein APRR3, suggesting an involvement in circadian rhythms (Murakami-Kojima et al., 2002; Nakamichi et al., 2002). Also, AtWNK8 interacts with subunit C of a vacuolar H+-ATPase, suggesting that this WNK might be involved in regulating ion transport in plants (HongHermesdorf et al., 2006). Our previous studies demonstrated that several AtWNK genes are involved in regulating the flowering time of Arabidopsis (Wang et al., 2008). However, no specific interacting partners of WNKs that are involved in hormonal regulation of development have been identified in plants. In this study, we have identified a new WNK homolog, GmWNK1, that is specifically expressed in soybean roots. We show that expression of GmWNK1 is predominantly detected in the root cells closely associated with lateral root formation, and its expression was dramatically influenced by ABA and multiple osmotic stresses. In vitro and in vivo experiments showed an interaction of GmWNK1 with GmCYP707A1, which is a key ABA 8¢-hydroxylase that functions in ABA catabolism. Alteration of GmWNK1 expression in transgenic soybean plants supported a role in the regulation of root system architecture, probably by finetuning ABA homeostasis via an ABA-dependent pathway. Taken together, our data demonstrate that GmWNK1 regulates root system architecture via an ABA-dependent pathway in the important food crop, soybean, and also suggest a new regulation pathway for ABA signaling in lateral root development. RESULTS Identification of GmWNK1 as a new root specific protein kinase in soybean A cDNA fragment designated GmWNK1 encoding a putative WNK kinase was identified by screening a soybean root cDNA library for root-specific genes expressed in response to abiotic stresses using the suppression subtraction hybridization technique (Guo et al., 2008). 5¢ and 3¢ RACE PCR was used to obtain the full-length GmWNK1 cDNA, which encodes a predicted protein with 610 amino acid residues and a calculated molecular weight of 70.15 kDa. Sequence analysis indicated that GmWNK1 is probably a bona fide WNK kinase due to its high degree of sequence similarity to other plant WNKs, including the defining characteristic of substitution of cysteine for lysine in kinase subdomain II (Figure S1a). The conserved regions of GmWNK1 share 62, 68 and 72% identity with WNK1 proteins

ª 2010 The Authors Journal compilation ª 2010 Blackwell Publishing Ltd, The Plant Journal, (2010), 64, 230–242

232 Yingxiang Wang et al. but not in the other tissues tested. We further investigated the expression of GmWNK1 within the root by dividing the root into three regions: part I from the primary root tip to the first visible lateral root, part II comprising the rest of the primary root except the lateral roots, and part III comprising the lateral roots only. The results showed that expression of GmWNK1 was significantly higher in part I compared to parts II and III (Figure 1b). In situ mRNA hybridization was then performed to localize GmWNK1 expression. When longitudinal and cross-sections of the lateral root primordia prior to the emergence of lateral roots were hybridized with an antisense GmWNK1 probe, hybridization signals were detected at high levels in all cells except epidermal cells (Figure 1c,d); signal was also strongly detected in pericycle cells of the mature lateral root and primary root (Figure 1e,h). A weak mRNA signal was detected in the epidermal cells of the primary root

from Arabidopsis, wheat and tobacco, respectively. The N-terminal serine/threonine kinase domain of GmWNK1 is similar to that of other plant and animal WNKs, i.e. it includes the 12 classical subdomains of the catalytic domain (Figure S1a). In addition to the kinase domain, another domain is conserved in plant WNKs and is not found in mammalian WNK family members. This has been designated the ‘PWNK’ (plant WNK) motif (Norihito et al., 2002; Xu et al., 2002), encompasses approximately 60 amino acids, and is located in the GmWNK1 C-terminal region (Figure S1a). According to a phylogenetic analysis, GmWNK1 belongs to group IA (Figure S1b). Expression pattern of GmWNK1 Real-time PCR was performed to examine the expression of GmWNK1 in various tissues. As shown in Figure 1(a), expression of GmWNK1 was specifically detected in roots

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Figure 1. Expression patterns for GmWNK1. (a) Real-time PCR analysis of GmWNK1 transcripts in various tissues of 60-day-old soybean seedlings. The soybean tubulin gene was used as an internal control. (b) Northern blot analysis of expression of GmWNK1 in various root regions. The root was divided into three regions: part I from the primary root tip to the first visible lateral root, part II comprising the rest of the primary root except the lateral roots, and part III comprising the lateral roots. Each lane contains 15 lg of total RNA extracted from the roots of 1-week-old soybean seedlings. The bottom panel shows the ethidium bromide-stained rRNA in the gel, indicating that an equal amount of RNA was loaded in each lane. (c–h) RNA in situ hybridization with a GmWNK1 probe. Scale bar = 200 lm. (c) Cross-section of the zone of lateral root development. (d) Longitudinal section of the zone of lateral root initiation. (e,h) Cross-sections of the zone before lateral root primodium differentiation. Endodermal (E), cortical (C) and epidermal (Ep) cell layers are shown in (e). (f,g) Negative controls with the sense GmWNK1 RNA probe. (i–l) Expression of pro:GmWNK1-GUS in transgenic soybean hairy roots. Scale bars = 10 mm (i), 20 lm (j) and 200 lm (k,l). The red and black arrows indicate protoxylem and protophloem, respectively, in (f) and (k).


GmWNK1 regulates root system architecture in soybean 233 (Figure 1h). A parallel experiment using the sense probe was performed as a control, showing only background signal (Figure 1f,g). To further characterize the expression pattern of GmWNK1, the upstream promoter sequence of GmWNK1 was amplified using high-efficiency thermal asymmetric interlaced PCR (hiTail-PCR) (Table S1), and was then fused to the GUS reporter gene. The chimeric gene cassette was introduced into soybean hairy roots. Histochemical staining for GUS activity in independent transgenic hairy roots showed that GmWNK1 was expressed predominantly in the vascular tissue of primary and lateral root primordia (Figure 1i,j), consistent with the RNA gel-blot results showing highest expression of GmWNK1 in part I of the roots (Figure 1b). Root cross-sections showed that GUS staining was observed in pericycle cells of the primary root, lateral root primordia, and all cells of younger lateral roots except epidermal cells (Figure 1k,l); these observations are also in agreement with the mRNA in situ hybridization results. GmWNK1 is down-regulated by ABA and osmotic stresses Several studies have reported that the RSA is profoundly affected by both intrinsic and environmental signals (Casimiro et al., 2003; Malamy, 2005; De Smet et al., 2006; Fukaki et al., 2007; Swarup et al., 2008). Therefore, we further examined the expression of GmWNK1 in response to various environmental stimuli. These include elemental stresses such as N, P, K and Fe deficiency and Al toxicity, various osmotic stressors such as NaCl, polyethylene glycol (PEG), mannitol, glucose and KCl, as well as the phytohormones ABA, indole-3-acetic acid (IAA); 6-benzyladenine (BA); gibberellic acid (GA). The results showed that the level of GmWNK1 transcript was significantly repressed by ABA and the osmotic stresses caused by PEG, NaCl, mannitol and glucose, but not by elemental stresses, including N, P, K and Fe deficiency and Al toxicity (Figure 2a) or use of other phytohormones (Figure S2). The effects of ABA and osmotic stimuli on GmWNK1 expression were stronger at 12 h than that at 2 h after treatment (Figure 2a). More detailed experiments were performed to investigate the temporal patterns of GmWNK1 expression in response to ABA, NaCl, KCl, PEG, mannitol and glucose. We found that repression of GmWNK1 by ABA started significantly later compared with the other treatments (Figure 2b). Specifically, GmWNK1 expression was significantly repressed at 9 h after treatment with ABA, but already repressed at just 2 h after treatment with NaCl and KCl, and at 6 h after treatment with PEG or mannitol (Figure 2b). Moreover, the relatively late response to ABA was also in agreement with the results from GUS staining in transgenic hairy roots carrying a GmWNK1–GUS fusion (Figure 2c). Sequence analysis of the promoter of GmWNK1 found that it contains several organ-/root-specific cis-elements and others asso-

ciated with drought-related ABA-responsive elements, which contain recognition sites for the MYB and MYC transcription factors (Table S2). GmWNK1 interacts with the ABA 8¢-hydroxylase GmCYP707A1 in vitro and in vivo GmWNK1 has two domains, an N-terminal catalytic domain and a C-terminal regulatory domain, which have distinct functions in mammals (Kahle et al., 2003, 2004). To discover potential upstream regulators and/or downstream targets of GmWNK1, a bacterial two-hybrid system was utilized to screen a soybean root cDNA library using the two domains of GmWNK1 as bait (Figure S3) (Joung et al., 2000; Wang et al., 2009). Ten positive transformants that showed interaction with the C-terminal end of GmWNK1 were identified. After sequence analysis, three clones were found to encode proteins with putative functions. One of these three was isolated using the C-terminal bait and is predicted to function in ABA metabolism, and was selected for further investigation. The full-length cDNA was isolated by 5¢ and 3¢ RACE, and the amino acid sequence of the GmWNK1-interacting protein was found to share 93 and 74% identity with ABA 8¢-hydroxylases from common bean and Arabidopsis, respectively. We named this gene GmCYP707A1. Multiple sequence alignments showed that GmCYP707A1 had the same conserved domains as P450 and Cypx (Figure S5a) (Krochko et al., 1998). Phylogenetic analysis revealed that GmCYP707A1 belongs to a class of genes that includes AtCYP707A1 and AtCYP707A3 in Arabidopsis (Figure S5b). Thus GmCYP707A1 is a putative ABA 8¢-hydroxylase from soybean. To further investigate the interaction of GmWNK1 with GmCYP707A1, we used GmWNK1 full-length, N- and C-terminal bait constructs (Figure S3). As expected (Figure 3a), the C-terminal region of GmWNK1 interacted with GmCYP707A1, but the N-terminal region did not, indicating that the C-terminal region is essential for the interaction between GmWNK1 and GmCYP707A1 in the bacterial two-hybrid system. We then tested the interaction between GmWNK1 and GmCYP707A1 in living cells using a bimolecular fluorescence complementation (BiFC) assay (Bracha-Drori et al., 2004). GmWNK1 and GmWNK1C were fused to N-terminal region of yellow fluorescent protein (YN), and GmCYP707A1 was fused to C-terminal yellow fluorescent protein (YC), and the fusion proteins were used for BiFC assays. When the fusions GmWNK1–YN or GmWNK1C–YN were co-expressed with GmCYP707A1–YC in soybean cell protoplasts, a yellow fluorescent signal was reproducibly detected in the cytoplasm compared to the controls (Figure 3b). Recent studies have shown that BiFC complexes formed by interaction partners produce higher fluorescence intensities that can be quantified by flow cytometry (Robida and Kerppola, 2009). We thus compared the fluorescence intensities following


234 Yingxiang Wang et al. Figure 2. Expression of GmWNK1 in response to various environmental stimuli. (a) Northern blot analysis of GmWNK1 expression patterns in response to various treatments. The roots of 11-day-old soybean seedlings grown in hydroponics were used for analysis. Details of the treatments are given in Experimental procedures. Total RNA (20 lg) isolated from the various treatments was hybridized with [a-32P]dCTP-labeled GmWNK1 DNA for 2 or 12 h. Ethidium bromide-stained gels (rRNA) are shown as a control for equal loading. (b) Northern blot analysis of GmWNK1 expression in response to ABA and other osmotic stresses over various time periods. Root samples were harvested at 0, 0.5, 2, 6, 9 and 12 h after treatment. CK indicates samples harvested before treatment. (c) Expression of proGmWNK1-GUS in transgenic soybean hairy roots in response to various stimuli. Scale bar = 10 mm.

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co-expression of the various interaction partners and their controls. The fluorescence intensities were higher when GmWNK1–YN or GmWNK1C–YN were co-expressed with GmCYP707A1–YC than when they were expressed with no interacting fusions (Figure S4). Together, these results support the idea that GmWNK1 and GmCYP707A1 do indeed interact in vivo. Our results on the interaction of full-length GmWNK1 with GmCYP707A1 also suggested that the N-terminal region does not interfere with interaction of the C-terminal region with GmCYP707A1. To further investigate the localization of GmWNK1 and GmCYP707A1, we fused cDNAs encoding GmWNK1 and GmCYP707A1 with that for green fluorescent protein (GFP), and placed these chimeric cDNAs downstream of the 35S promoter. The constructs were introduced into onion epidermal cells by particle bombardment. Free GFP was expressed strongly in the nucleoplasm, as well as in the cytoplasm and the plasma membrane (Figure 4). In contrast,

the GmWNK1–GFP and GmCYP707A1–GFP fusion proteins were observed with similar patterns in the cytoplasm, including filamentous and some punctate patterns (Figure 4), consistent with their probable physical interaction. GmCYP707A1 expression is up-regulated by ABA and osmotic stresses Similar to GmWNK1, GmCYP707A1 was expressed in roots but not in shoots (Figure S6). In addition, GmCYP707A1 expression was specifically induced by ABA (Figure 5a). A time-course experiment was also performed to investigate the temporal patterns of GmCYP707A1 expression in response to ABA, NaCl, KCl, PEG, mannitol and glucose. The transcript level of GmCYP707A1 was enhanced by ABA treatment, reaching a peak at 6 h, and then remaining at a high level. Furthermore, its expression was induced by PEG and mannitol, peaking within 30 min and then gradually decreased to very low levels and fully disappearing at 6 h


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Figure 3. GmWNK1 interacts with GmCYP707A1 both in vitro and in vivo. (a) Interactions between GmCYP707A1 and each construct were determined by bacterial growth assay on medium lacking 3-AT and in the presence of 5 mM 3-amino-1,2,4-triazole (3-AT). The blue color indicates b-galactosidase activity and confirms the positive interactions. (b) Detection of GmWNK1–GmCYP707A1 interactions in soybean protoplasts by BiFC. Cells were observed on a confocal microscope under bright field (left), YFP fluorescence (middle) and merged (right). Scale bars = 10 lm.

after treatment with mannitol. These findings imply that, in response to PEG and mannitol, there is an increase in endogenous ABA levels, which then enhance GmCYP707A1 expression. The effects of KCl and glucose on GmCYP707A1 expression were slower than those of PEG and mannitol (Figure 5b). These results indicate that GmCYP707A1 responded differently to ABA, NaCl, KCl, PEG, mannitol and glucose, and its expression pattern was almost opposite to that of GmWNK1 in response to ABA and osmotic stress. Over-expressing GmWNK1 changes the root system architecture in transgenic soybean plants To investigate the in vivo functions of GmWNK1 in regulation of root development in soybean, we generated stable transgenic soybean plants over-expressing GmWNK1 using Agrobacterium tumefaciens-mediated transformation of the soybean cotyledon node. Homozygous T3 plants of two independent single-copy transgenic lines (GmWNK1-OE1 and GmWNK1-OE2) were characterized for root traits compared with wild-type plants. The transgenic lines were analyzed by genomic DNA hybridization (Figure 6a), resistance to herbicide (Figure 6b) and real-time PCR analysis of GmWNK1 expression compared to wild-type (Figure 6c). The transgenic soybean plants over-expressing GmWNK1 were smaller in size compared with their non-transgenic segregants during all growth stages studied (Figure 6d,e)

and had other traits, including altered total root length and lateral root number, particularly in response to ABA (Figure 6f–i). Previous reports have shown that endogenous ABA levels are increased by osmotic stressors such as mannitol, sorbitol and PEG in plants (Jia et al., 2002; Chinnusamy et al., 2008). Therefore, the endogenous levels of ABA could differ in the transgenic plants relative to control plants, as suggested by the phenotypic characterization. To test this hypothesis, endogenous ABA levels were determined in the transgenic plants and their non-transgenic segregants with or without ABA treatment. Endogenous ABA levels in the transgenic plants were significantly increased by approximately two- and fivefolds compared to the non-transgenic controls, with or without ABA treatment, respectively (Figure 7a). We also used GmABI1, a homolog of AtABI1 (encoding a protein phosphatase 2C), which is an indicator of ABA transduction signals in Arabidopsis (Merlot et al., 2001), to monitor the endogenous ABA levels in transgenic plants with or without ABA treatments. As shown in Figure 7(b), the GmABI1 transcript level was significantly increased in the transgenic plants compared with their control lines. These results further demonstrate the potential role of GmWNK1 in regulating lateral root development by modulating endogenous ABA levels or ABA signaling in soybean.


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Figure 4. Subcellular localization of GmWNK1 and GmCYP707A1. Cells expressing GFP (control) (a,b), GmWNK1– GFP (c,d) and GmCYP707A1–GFP (e,f) under the control of the CaMV 35S promoter. Scale bar = 50 lm.

Figure 5. Expression patterns of GmCYP707A1 under various treatments. (a) Northern blot analysis of GmCYP707A1 expression in response to various phytohormones. The roots of 11-day-old soybean seedlings were used, and the method used was as described for GmWNK1 analysis. (b) Northern blot analysis of GmCYP707A1 expression over various time periods. The same samples were used as for GmWNK1 analysis.

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Figure 6. Over-expression of GmWNK1 regulates lateral root development in trangenic soybean plants. (a) Southern blot analysis of the insertion in the two transformed lines (GmWNK1-OE1 and GmWNK1-OE2) using the bar gene as a probe. DNA was digested by EcoRI (RI) or HindIII (HIII), respectively. M, molecular marker of k DNA digested by HindIII; W, wild-type; 1, GmWNK1-OE1; 2, GmWNK1-OE2. (b) Confirmation of T3 transgenic plants by herbicide resistance. Left, non-transgenic segregant from transgenic event; right, over-expressing line. Scale bar = 1 cm. (c) Real-time PCR analysis of the expression of GmWNK1 in transgenic plants and wild-type. Values are the mean of three independent biological replicates SE. (d) Phenotype of 1-week-old T3 transgenic soybean plants and wild-type with ABA (100 lM) and without ABA. Scale bar = 4 cm. (e) Phenotype of 35-day-old T3 transgenic soybean plants and wild-type in hydroponics under normal conditions. Scale bar = 4 cm. (f–i) Phenotypic characterization of the roots from two over-expressing GmWNK1 and control lines with ABA (100 lM) and without ABA. Plants were grown in hydroponics for 10 days. Values are the mean of three independent biological replicates SE (**P < 0.01 versus wild-type).

DISCUSSION GmWNK1 is a new member of the WNK kinase subfamily in soybean The WNK gene family has been characterized as a new subfamily of serine/threonine protein kinases in mammals (Xu et al., 2002), which have several characteristic features. First, WNK kinases have the common feature of all serine/ threonine kinases of a catalytic domain of approximately 300 amino acid residues that can be subdivided into 12 domains, each with several highly conserved residues (Xu et al., 2002; Wang et al., 2008). Second, in serine/threonine kinases, one

of these conserved residues is a lysine (K) located in subdomain II of the kinase domain. This K residue is critical for ATP binding at the catalytic site, but is replaced by other amino acids in WNKs. Third, another lysine residue in subdomain I is conserved in WNKs, and provides the function of the missing lysine in subdomain II. Fourth, WNKs have a conserved region called the autoinhibitory domain, which is C-terminal to the catalytic domain and has been shown to modulate the kinase activity in vitro (Xu et al., 2002; Wang et al., 2008). Our recent studies showed that all the above properties of mammalian WNKs were found in flowering plants, suggesting that WNK catalytic properties are


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Figure 7. Endogenous ABA content and expression of GmABI1 in 35S-GmWNK1 transgenic plants and wild-type. (a) One-week-old seedlings of wild-type and 35S-GmWNK1 transgenic plants were grown in hydroponics for ABA quantification by ELISA with ABA (100 lM) and without ABA. Values are the mean of three independent biological replicates SE (n = 3, **P < 0.01). FW, fresh weight. (b) Expression of the ABA-responsive gene GmABI1 was determined by real-time PCR. GmEF1a was used as an internal control. WT, wild-type; OE1, over-expression line 1; CK, normal conditions; ABA, 100 lM.

conserved between animals and plants (Wang et al., 2008). The present study identified a new protein GmWNK1 based on the four characteristics of WNKs mentioned above (Figure S1a). Mammals such as human and mouse each have four WNK genes, each with several highly conserved residues (Xu et al., 2002). By contrast, plants have a larger number of WNK genes (Nakamichi et al., 2002; Hong-Hermesdorf et al., 2006; Wang et al., 2008). Our previous phylogenetic analysis of WNKs showed that plant and animal WNKs form very well-supported kingdom-specific clades, without any clades that contain both plant and animal genes, suggesting that they have evolved separately since the divergence of plants and animals from their most recent common ancestor (Wang et al., 2008). Plant WNKs can be further divided into four subgroups, indicating that the plant WNK gene family has experienced dynamic evolution during angiosperm evolution. The various evolutionary patterns may have resulted in adaptive changes in function. GmWNK1 belongs to subgroup I (Figure S1b). Taken together, these results strongly support our contention that GmWNK1 is a WNK kinase of soybean. Interactions between GmWNK1 and GmCYP707A1 in regulation of the ABA pathway The recent discovery of genes involved in ABA catabolism has provided new insights into the regulation of ABAmediated responses. Although several alternative catabolic pathways may exist for the inactivation of ABA, hydroxylation of ABA at the 8¢ position to produce 8¢-hydroxyl ABA appears to be the most prominent (Nambara and MarionPoll, 2005). The CYP707A family of cytochrome P450 proteins is known to encode an ABA 8¢-hydroxylase in plants (Kushiro et al., 2004). The main function of this gene family is to catalyze the first committed step in the ABA catabolic pathway (Krochko et al., 1998). The CYP707A gene family is highly conserved across plant species (Kushiro et al., 2004; Millar et al., 2006; Yang and Zeevaart, 2006; Saika et al.,

2007). However, the regulation and mechanistic function of CYP707A are still unclear. Consistent with regulation of its expression by ABA-associated responses, we used the bacterial two-hybrid system and the in vivo BiFC system to show that GmWNK1 interacts with a key enzyme of ABA metabolism, the C-terminal region of the 8¢-hydroxylase GmCYP707A1 (Figure 3). The similar tissue expression and subcellular localization patterns of GmCYP707A1 and GmWNK1 in soybean roots support the physiological relevance of the interactions between these two proteins. Previous studies have shown that the various domains of WNK kinases have distinct functions on multiple targets in animals (Kahle et al., 2003, 2004; Yang et al., 2005, 2007). In Arabidopsis, the C-terminal domain of AtWNK8 binds to AtVHA-C, suggesting that this region represents a direct interaction module that facilitates the specific and efficient phosphorylation of subunit C of V-ATPase (Hong-Hermesdorf et al., 2006). We also found that the subcellular localization of AtWNK8 was similar to that of GmWNK1 (Wang et al., unpublished data), indicating that the main targets of WNKs could be some of the proteins present in the inner membranes of plants. Here, the in vivo function of GmWNK1 was studied via gain-of-function analysis in whole transgenic soybean plants, and transgenic over-expression of GmWNK1 in soybean was found to significantly affect the root development as indicated by reductions in total root length, lateral root number and length (Figure 6). This phenomenon was especially evident in response to ABA treatment (Figure 6). Moreover, endogenous ABA levels were also found to be higher in transgenic plants compared with wild-type plants under normal and ABA treatment conditions (Figure 7a). The expression pattern of GmABI1 was also affected in overexpressing GmWNK1 soybean plants compared with control lines (Figure 7b). Taken together, our results support the proposition that GmWNK1 is indeed involved in regulation of the root system architecture via an ABA-associated pathway.


GmWNK1 regulates root system architecture in soybean 239 Roles of GmWNK1 in regulation of the RSA The specific expression of GmWNK1 in soybean roots, particularly in the root regions related to lateral root initiation and development (Figure 1), strongly suggests that the GmWNK1 gene might be involved in regulation of lateral root development in soybean. Previous studies indicated that the plasticity of the RSA is profoundly affected by many components of intrinsic and environmental response pathways (Malamy, 2005; De Smet et al., 2006). Here, the responses of GmWNK1 to various stimuli indicated that GmWNK1 expression was significantly affected by ABA and its associated osmotic stresses (Figure 2a–c). This finding is generally similar to those for mammalian WNKs, which are also regulated by hyperosmotic stress, affecting ion homeostasis and leading to hypertension or hyperkalemia (Wilson et al., 2001; Zagorska et al., 2007). The similarity between WNK function in mammalian osmotic processes and the role of ABA in regulating GmWNK1 is intriguing. ABA plays critical roles in many important plant developmental and physiological processes, including seed development, seed dormancy and adaptation to adverse environmental stresses such as osmotic stress in saline soils (Hasegawa et al., 2000; Verslues and Zhu, 2005; Chinnusamy et al., 2008). ABA has also been strongly implicated in regulating lateral root formation in response to environmental cues (Malamy, 2005; De Smet et al., 2006). The combination of the tissue and cell expression patterns of GmWNK1 in the soybean root and the role of GmWNK1 in ABA pathways confirms that we have discovered a homolog of animal WNKs that regulates the RSA in response to environmental conditions in an ABA-dependent fashion. Given the response of GmCYP707A1 and GmWNK1 to ABA-associated pathways and their direct physical interaction, as well as their putative physiological functions, we propose a speculative model in which GmWNK1 acts as a negative regulator of GmCYP707A1 in the ABA-dependent regulation of lateral root development (Figure S7). In this model, osmotic stress increases the endogenous ABA level, which then represses the expression of GmWNK1, reducing or releasing its inhibition of GmCYP707A1 in ABA catabolism. Possibly, these regulatory steps form a feedback loop to help fine-tune ABA homeostasis in vivo, and then influence lateral root development. The number and position of lateral roots are a key determinant of plant survival and crop yield, particularly under water, nutrient or other stress conditions. Therefore, understanding the regulatory mechanisms that control the RSA in response to environmental cues has important applications in agriculture. Because WNK kinases have been identified as functioning in mammalian cells (Anselmo et al., 2006), amphibian oocytes (Gagnon et al., 2006), nematodes (Choe and Strange, 2007) and now plant cells in response to osmotic stress, it seems likely that WNKs arose early in evolution to

provide both plant and animal cells with a homeostatic counter-response to combat the potent environmental challenges that threaten cell volume integrity. Future molecular, genetic and physiological studies that help to elucidate the interaction of GmWNK1 and GmCYP707A1 might assist in improving crop tolerance to specific abiotic stresses. In summary, these findings suggest that GmWNK1 might play an important role in the ABA-mediated homeostatic responses by which soybean roots deal with osmotic challenges in the soil. EXPERIMENTAL PROCEDURES Plant materials and growth conditions A soybean (Glycine max L. Merr.) cultivar, HN89, was used in this study. Soybean seedlings were cultured in modified half-strength Hoagland nutrient solution in hydroponic culture under a 12 h/12 h light/dark regimen at 28C, with a light intensity of 500 lmol photons m)2 sec)1. For various treatments, after 10 days of growth under normal conditions, the plants were subjected to various stresses: 200 mM NaCl, 15% PEG6000, 150 lM ABA, deficiency of P (10 lM), N (0 lM), K (0 lM) or Fe (0 lM), excess Al (316 lM), excess high phosphate (HP) (320 lM), 30 lM IAA, 100 lM 2,3,5-triiodobenzoic acid (TIBA), 15 lM GA, 100 lM 6-BA, 10% glucose or 200 mM mannitol, respectively.

Isolation of GmWNK1 cDNA by suppression subtraction hybridization (SSH) A Clontech PCR-Select cDNA subtraction kit (http://www.clontech. com/) was used to construct the soybean root subtractive library (see Appendix S1). The tester and driver cDNAs were originated from the roots and leaves of soybean, respectively. The full-length cDNA sequence of GmWNK1 was obtained by 5¢ and 3¢ RACE techniques using specific primers (Table S3) and a SMART RACE cDNA amplification kit (Clontech). The fragments obtained were subcloned into the pGEM-T Easy vector (Promega, http:// www.promega.com/) for sequencing.

Cloning of the promoter of GmWNK1 using hiTAIL-PCR The primers used for promoter cloning (Table S4) and the experimental procedures were based on the hiTAIL-PCR technique described by Liu and Chen (2008) (see details in Appendix S1). The PCR amplification procedure, with slight modification of the temperature, is shown in Table S1. The fragments obtained were subcloned into the pGEM-T Easy vector (Promega) for sequencing. The transcription start sites of GmWNK1 were predicted using the Neural Network Promoter Prediction of the Berkeley Drosophila Genome Project (http://www.fruitfly.org/seq_tools/promoter.html). The promoter cis-elements were predicted using PLACE software (http://www.dna.affrc.go.jp/PLACE).

Real-time PCR and in situ hybridization Plant tissue was collected and quickly frozen in liquid nitrogen. Total RNA was extracted using Trizol reagent (Invitrogen, http://www. invitrogen.com/). Approximately 1 lg total RNA was used for reverse transcription according to the manufacturer’s instructions (Promega). Real-time PCR was performed using Brilliant SYBR Green QPCR Master Mix (Takara, http://www.takara-bio.com/) and an ABI One-step platform (Applied Biosystems, http://www. appliedbiosystems.com/). Each sample was analyzed in triplicate.


240 Yingxiang Wang et al. The data were normalized using the reference gene GmEF1a, and the relative mRNA amount was calculated using the comparative Ct method. Primer sets used are listed in Table S3. Non-radioactive RNA in situ hybridization was performed as described previously (Li et al., 2004). A 3’ terminal GmWNK1 cDNA fragment of 500 bp was amplified using GmWNK1-specific primers (Table S3). The PCR product was confirmed by sequencing, and cloned into the pGEM-T vector (Promega) to yield a plasmid that was completely digested by SpeI or NcoI and used as template for transcription with SP6 or T7 RNA polymerases, respectively (Roche, http://www.roche.com). Images were obtained using an Olympus BX-51 microscope (http://www.olympus-global.com/) with a SPOT II RT camera (Diagnostic Instruments, http://www.diaginc.com), and edited using Photoshop CS2 (Adobe, http://www.adobe.com/).

Bimolecular fluorescence complementation BiFC vectors were constructed using N-terminal YFP (YFPN, amino acids 1–155) and C-terminal YFP (YFPC, amino acids 156–239) vectors (Bracha-Drori et al., 2004). GmWNK1 and GmWNK1C were amplified using primer pairs BiFC-GmWNK1F/BiFC-GmWNK1R and BiFC-GmWNK2F/BiFC-GmWNK1R, and ligated into the XbaI/KpnI sites of YFPN, while GmCYP707A1 was amplified using primer pair BiFC-Gm CYP707A1F/BiFC-Gm CYP707A1R and ligated into the XbaI/KpnI sites of YFPC (see Table S3). A control vector comprising GFP transcribed from the CaMV 35S promoter in a pUC18-based vector was obtained as described previously (Wang et al., 2006). Soybean protoplasts were isolated from soybean suspension cells by digestion at 25C in the dark using 0.2% Pectolyase Y-23 and 0.5% Cellulase RS (Yakult Biochemicals, http://www.yakult.co.jp). The protoplasts were washed in 4 mM MES buffer containing 0.5 M mannitol and 20 mM KCl, and were transfected using a modified polyethylene glycol method as described previously (Kovtun et al., 2000). The transfected protoplasts were incubated at 25C for 10 h, and YFP fluorescence was observed using a Leica TCS 4D confocal laser scanning microscope (Leica, http://www.leica.com/).

Soybean stable transformation The 35S-GmWNK1 plasmid was introduced into A. tumefaciens strain EHA101 by the freeze–thaw method. The cotyledonary-node method as described by Paz et al. (2004) was used with the following modifications to improve the transformation efficiency. Transformants were selected on 3.5 mg L)1 glufosinate for 4 weeks after shoot initiation, followed by an additional 4–6 weeks under 2.5 mg L)1 glufosinate selection for shoot elongation (see details in Appendix S1). Primary positive transformants identified by herbicide resistance screening, DNA gel blot and RNA analyses were further analyzed. Root phenotypic assays were performed using T3 homozygous lines and the control (separated from the transformation events).

Endogenous ABA measurement One-week-old seedlings of transgenic plants and wild-type were used for quantitative determination of endogenous ABA concentration using a Phytodetek ABA test kit (Sigma, http://www.sigmaaldrich.com/). The harvested samples were immediately ground into fine powder on ice, and then exposed to extraction buffer (80% v/v aqueous methanol plus 100 mg L)1 butyrate hydroxytoluene) at 4C for 4 h. The samples were centrifuged at 4000 g for 15 min, and the residues were re-extracted with extraction buffer for 1 h. The supernatant was then purified using Sep-Pak C18 cartridges (Waters Associates, http://www.waterassociates.com). The combined methanol extracts were evaporated almost to dryness under reduced pressure at 4C, and dissolved in 300 ll of TBS buffer

(Tris-buffered saline: 150 mM NaCl, 1 mM MgCl2 and 50 mM Tris, pH7.8) for ELISA analysis.

ACKNOWLEDGEMENTS This work was supported by grants from the National Natural Science Foundation of China (Grant No. 30900918) to Y. W., the National Key Basic Research Special Funds of China (2005CB120902) and the McKnight Foundation’s Collaborative Crop Research Program (05-780) to X. Y. We are grateful to Dr. Chengbin Xiang (School of Life Sciences, University of Science and Technology of China) for providing the pDOR207 and p308R vectors and valuable comments on this manuscript, Dr. Tom Kerppola (Department of Biological Chemistry Investigator Howard Hughes Medical Institute) for the YFP vector. We are also grateful to Dr. Yongchao Liang (Chinese Academy of Agriculture Science at Beijing), Dr. Tom Beeckman (Plant Systems Biology, Ghent University of Belgium), Dr. Leon Kochian (Department of Agriculture-Agricultural Research Service at Cornell University) for the critical review of this manuscript.

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Multiple sequence alignments and phylogenetic relationship of GmWNK1. Figure S2. Expression of GmWNK1 in response to various phytohormones. Figure S3. Various constructs of GmWNK1. Figure S4. Quantification of the fluorescence intensity of BiFC by flow cytometry. Figure S5. Phylogenetic analysis of GmCYP707A1. Figure S6. Expression pattern of GmCYP707A1 in the soybean seedling. Figure S7. Model for role of GmWNK1 in regulation of the root system architecture. Table S1. Thermal conditions for hiTAIL-PCR. Table S2. cis-elements in the promoter of GmWNK1. Table S3. Gene-specific primers used in this study. Table S4. Primers used for GmWNK1 promoter cloning and construction. Appendix S1. Additional experimental procedures. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

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The Genbank accession numbers for the GmWNK1 and GmCYP707A1 sequences are ABQ65855 and ABQ65856, respectively.
