Overexpression of the soybean GmWNK1 altered the sensitivity to salt and osmotic stress in Arabidopsis

Journal of Plant Physiology 168 (2011) 2260–2267

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Overexpression of the soybean GmWNK1 altered the sensitivity to salt and osmotic stress in Arabidopsis Yingxiang Wang a,b,∗ , Haicui Suo a , Chuxiong Zhuang c , Hong Ma b,d,e , Xiaolong Yan a,1 a

Root Biology Center, South China Agricultural University, Guangzhou 510642, PR China State Key Laboratory of Genetic Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, PR China Department of Biology, South China Agricultural University, Guangzhou 510642, PR China d Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, PR China e Department of Biology, The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, 16802 PA, USA b c

a r t i c l e

i n f o

Article history: Received 6 September 2010 Received in revised form 8 July 2011 Accepted 11 July 2011 Keywords: GmWNK1 Soybean Arabidopsis Osmotic stresses Transgene

s u m m a r y The WNK (With No Lysine K) serine–threonine kinases have been shown to be osmosensitive regulators and are critical for cell volume homeostasis in humans. We previously identified a soybean root-specific WNK homolog, GmWNK1, which is important for normal late root development by fine-tuning regulation of ABA levels. However, the functions of WNKs in plant osmotic stress response remains uncertain. In this study, we generated transgenic Arabidopsis plants with constitutive expression of GmWNK1. We found that these transgenic plants had increased endogenous ABA levels and altered expression of ABAresponsive genes, and exhibited a significantly enhanced tolerance to NaCl and osmotic stresses during seed germination and seedling development. These findings suggest that, in addition to regulating root development, GmWNK1 also regulates ABA-responsive gene expression and/or interacts with other stress related signals, thereby modulating osmotic stress responses. Thus, these results suggest that WNKs are members of an evolutionarily conserved kinase family that modulates cellular response to osmotic stresses in both animal and plants. © 2011 Elsevier GmbH. All rights reserved.

Introduction The WNK serine–threonine kinases have the unique characteristic of an atypical placement of the catalytic lysine in sub-domain II and was first discovered through screening genes essential for the human autosomal dominant disease of salt-sensitive hypertension, Pseudohypoaldosteronism type II (Wilson et al., 2001). In humans, mutations in either WNK1 or WNK4 cause hypertension by increasing the reabsorption of NaCl across epithelia in distal nephrons of the kidney (Kahle et al., 2003). Subsequent analyses show that other members of the WNK family are osmosensitive regulators of ion transport: they regulate a diverse array of ion transporters and channels and are critical for the homeostasis of cell volume and epithelial ion transport (Kahle et al., 2008). Besides the wellestablished roles in the regulation of electrolyte flux in animals, they have also been implicated in several other processes, including cell growth, differentiation, apoptosis, and development (Kahle et al., 2006).

∗ Corresponding author at: Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, PR China. Fax: +86 21 55664187. E-mail addresses: yx [email protected], [email protected] (Y. Wang). 1 Deceased. 0176-1617/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2011.07.014

The WNK homologs have also been identified in other species, including 10 Arabidopsis members, which play diverse roles in flowering time, regulation of circadian rhythms and interaction with V-ATPase (Murakami-Kojima et al., 2002; Nakamichi et al., 2002; Hong-Hermesdorf et al., 2006; Wang et al., 2008; Tsuchiya and Eulgem, 2010). More recently, a soybean root-specific WNK homolog, GmWNK1, had been identified to play a critical function in regulating ABA homeostasis for normal root system architecture (Wang et al., 2010). In comparison to their diverse physiological functions in animals, functions of plant WNK kinases are very limited. To better address the function of GmWNK1 in plants, fusion of a GmWNK1 full-length cDNA of GmWNK1 driven by the CaMV 35S promoter was introduced into the model plant of Arabidopsis, which is an excellent system for physiological characterization due to its many advantages in molecular analysis over transgenic soybean. Here, we reported the investigation of the tolerance of 35S-GmWNK1 transgenic plants in response to ABA or other osmotic stresses. We also examined the expression patterns of stress-related genes in both 35S-GmWNK1 transgenic plants and wild type under stress conditions. The results showed that overexpression of GmWNK1 in Arabidopsis increased endogenous levels of ABA, altered the expression of known ABA-responsive genes, and enhanced tolerance to NaCl and other osmotic stresses during early

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vegetative development. Our data provide evidence that WNKs are evolutionarily conserved regulators of cellular response to osmotic stress. Materials and methods Plant growth conditions and morphological analysis Arabidopsis (ecotype Columbia) plants were grown in growth chambers at 22 ◦ C/18 ◦ C under a 16-h-light/8-h-dark photoperiod unless otherwise indicated. Five-day-old plants grown on half strength Murashige and Skoog (MS) medium were transferred for an additional 7 days or more on 1/2 MS medium supplemented with ABA, NaCl or mannitol of different concentrations as indicated in the text. Then the plants were photographed and root parameters were analyzed using Micro software of Image J. Plasmid construction and Arabidopsis transformation A full length GmWNK1 cDNA was amplified using a pair of specific primers (Forward: 5 -ACCTTATTGCGGGACAACAC-3 , Reverse: 5 -CCTTATGGAGA TTGATGGA G-3 ), and was subcloned into the pGEM-T vector (Promega), and verified by sequencing. The cDNA was then cloned into the final plasmid of pCAMBIA1380 downstream of the 35S promoter. Transformation of Arabidopsis was performed using the vacuum infiltration method (Bechtold and Pelletier, 1998). After selection on plates with 1/2 Murashige and Skoog medium containing 50 ␮g/L hygromycin. T1 positive plants were transplanted to soil, grown to maturity and their seeds harvested; T2 transgenic lines were then used for detailed phenotypic investigation. Transgenic Arabidopsis thaliana (ecotype Columbia) seeds were surface sterilized with 70% ethanol for 10 min and then with 10% household bleach for 5 min before being washed five times with sterilized water. The sterilized seeds were sown on agar plates containing 1/2 MS salts, vitamins, 0.3% phytogel (Sigma, USA), and 0.5% sucrose.

Fig. 1. Molecular characterization of 35S-GmWNK1 transgenic plants. Analysis of the GmWNK1 expression level in different transgenic lines using semi-quantitative RT-PCR. The two representative lines highlighted by an asterisk were used for further analysis. Col: wild type (ecotype Columbia) plants; Quantification of GmWNK1 expression in two representative lines by real time PCR. Col, wild type; T13 and T16 represent two different transgenic lines. The data were normalized using a reference EF1␣ (elongation factor 1␣: At1g07920). Error bars indicate standard errors (n = 3).

ACGAACA-3 , Reverse: 5 -GGCAATCCATTCTGAGGAAG-3 ). Gene specificity was verified for the CYP707A genes by sequencing. The other primers were used according to the reference (Miura et al., 2007). Endogenous ABA measurement Two-week-old seedlings of 35S-GmWNK1 transgenic plants and wild type Arabidopsis plants were grown under normal condition, and then were subjected to different stresses for another 48 h. Seedling were harvested for ABA contents analysis using the phytodetek ABA test kit (Catalog number: PDK 09347/0096, Sigma, USA) as described previously (Wang et al., 2010).

RNA analysis Results Total RNA was extracted using the Trizol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA). The first-strand cDNA was synthesized from 1 ␮g total RNA with oligo(dT) primer using Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA). Quantitative real-time RT-PCR was performed using the ToYoBo QPCR SYBR Green Mix kit (ToYoBo, Tokyo, Japan), in a Corbett Research Rotor-Gene 2000 cycler as described previously (Wang et al., 2008). Primers used to analyze expression of GmWNK1 were described by Wang et al. (2010). The others used were as follows: AtEF1? (At1g07920, Forward: 5 -AGCATACACTGCGTGCAAAG-3 , Reverse: 5 -TCGCCTGTGTCACATATCTC-3 ), RD29A (At5g52310, Forward: 5 -ATCACT TGGCTCCACTGTTG TTC-3 , Reverse: 5 -ACAAAACACACATAAACATC  CAAAGT-3 ), RD29B (At5g52300, Forward: 5 -GTGAAGATGACTATCTCGGTGGTC-3 , Reverse: 5 -C CTAACTCTCCGGTGTAACCTAG-3 ), RAB18 (At5g66400, Forward: 5 -CAGCAGCAGT ATGACGAGTA-3 , Reverse: 5 -CAGTTCCAAAGCCTTCAGTC-3 ), ABI1 (At4g26080, Forward:5 -AGAGTGTGCCTTTGTATGGTTTTA-3 , Reverse: 5 CAATAGTTCGCT-3 ), COR47 (At1g20440, CATCCTCTCTCTA Forward: 5 -GAGAAGCTTCCTGGT CACCA-3 , Reverse: 5 -CTTGGCATGATAACCTGGAAG-3) and KIN1 (At5g15960, Forward: 5 ACCAACAAGAATGCCTTCC A-3 , Reverse: 5 -CCGCATCCGATACACT CTTT-3 ), AtCYP707A1 (Forward: 5 -CAT TTGGCAATGGAACCCAC-3 , Reverse: 5 -GAA GTCTCAATCCAGGAGGA-3 ), AtCYP707A2 (Forward: 5 -CCGCTTCTGTCTTAA CTTGG-3 , Reverse: 5 -GGTAAACCCTTCTTGGGTAC-3 ), AtCYP707A3 (Forward: 5 -GCAGGATTAACCG-

Constitutive expression of GmWNK1 in Arabidopsis reduced ABA sensitivity in seed germination and seedling development We previously identified a soybean root specific protein kinase of GmWNK1 that was involved in lateral root development by regulating ABA catabolism (Wang et al., 2010). To investigate additional functions of GmWNK1 in vivo, we took advantage of the relative ease in generating and analyzing Arabidopsis transgenic plants. GmWNK1 expression in transgenic plants were detected by semi-quantitative and real-time PCR, two representative lines with relative higher expression level (named GmWNK1OX-1 and GmWNK1OX-2) were selected for further analysis (Fig. 1A and B). Our previous findings demonstrated that GmWNK1 expression was altered by multiple ABA associated stresses (Wang et al., 2010). This motivates us to test whether constitutive expression of GmWNK1 affects ABA sensitivity in Arabidopsis. We first examined seed germination due to its high sensitivity to ABA. Compared to wild type, 35S-GmWNK1 transgenic seeds germinated earlier under normal conditions (Fig. 2A). Thirty-six hours after planting, 49% of the 35S-GmWNK1 transgenic seeds germinated, whereas only 38% of wild type seeds did (Fig. 2A). In the presence of ABA, the germination rate of wild-type seeds decreased with increasing ABA concentration. 0.5 ␮M ABA was sufficient to decrease the germination of wild-type seeds to 40% of the untreated level, whereas the 35S-GmWNK1 transgenic seeds

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Fig. 2. Determination of germination rate for 35S-GmWNK1 transgenic seeds. Germination rate of 35S-GmWNK1 transgenic seeds on ABA-free medium and on media with different concentrations of ABA, NaCl or mannitol. Seeds of wild type, abi4-1, aba2-1 and two lines of transgenic plants were sown on plates for 4 days (fully emerged radicle) before being scored for germination. Each datum point represents the mean of triplicate experiments (n = 150).

retained approximately 80% germination rates under the same conditions (Figs. 2B and 3A–C). Furthermore, we compared the germination rate between 35S-GmWNK1 transgenic seeds and ABAdeficient (aba) or ABA-insensitive (abi) mutants (aba2-1 and abi4-1) (Leung et al., 1997; Merlot et al., 2001) in media containing various concentrations of ABA (Figs. 2B and 3D and E). As shown in Fig. 2B, over-expression of GmWNK1 conferred an intermediate level of germination rate and resistance to ABA between abi4-1 and wild type (or aba2). We then examined the root length of transgenic seedlings. Similar to the ABA response in seed germination rate, the 35S-GmWNK1 transgenic plants had lower ABA sensitivity for primary root growth than wild type at various ABA concentrations (Fig. 4B and C). Specifically, in the presence of 0.25 ␮M ABA, the average primary root length of wild type was 37.5% compared to control conditions, while the root lengths of the two 35S-GmWNK1 transgenic lines were 49% and 52.4%, respectively, of the untreated plants (Fig. 4B and C). At the higher concentration of 0.5 ␮M ABA, the relative primary root length was reduced to 11.2%, 21.6% and 23.2% for wild type and two transgenic lines, respectively (Fig. 4B and C). These results indicated that over-expression of the GmWNK1 gene in Arabidopsis thaliana endowed the plant with greater tolerance to exogenous ABA.

35S-GmWNK1 seedlings exhibit resistance to salt and osmotic stresses There is substantial evidence for cross-talk between signaling pathways regulating response to ABA and assorted stresses (e.g. drought, salinity and cold) (Finkelstein et al., 2002; Xiong et al., 2002). Typically, aba and abi mutants tend to exhibit salt insensi-

tivity during germination (Léon-Kloosterziel et al., 1996). Mannitol was frequently used to provide osmotic stress to plants (Patonnier et al., 1999). We therefore examined the germination responses of 35S-GmWNK1 transgenic, wild type and the abi4-1 and aba21seeds exposed to a range of concentrations of NaCl (Figs. 2C and 3F–J). In the presence of 200 mM NaCl, the germination rate of the wild-type was 43%, while those of the two 35S-GmWNK1 transgenic lines were 70% and 75%, respectively (n > 100). In the presence of 150 mM NaCl, wild-type root growth rate was only 12.2% of its control rate, whereas those of the two 35S-GmWNK1 transgenic lines were 27.1% and 27.5% of the control rates respectively (Fig. 4D and E). In a parallel experiment, the 35S-GmWNK1 transgenic lines responded to mannitol in a similar manner. At 300 mM mannitol, the germination rate of wild-type reached 48%, whereas germination rates for the two 35S-GmWNK1 transgenic lines were 76% and 75%, respectively (n > 100) (Figs. 2D and 3K–O). Under the same conditions, wild-type root growth rate was only 17.8% of its control rate, whereas those of the two 35S-GmWNK1 lines were 32.2% and 29.6% of the control rates, respectively (Fig. 4F and G). Taken together, these findings demonstrated that constitutive expression of GmWNK1 in transgenic plants reduced sensitivity to salt and mannitol stresses.

Expression of ABA responsive genes were altered in 35S-GmWNK1 transgenic plants We further examined whether the changes in sensitivity to ABA or related osmotic stresses in 35S-GmWNK1 transgenic plants were accompanied by altered expression of ABA-responsive genes,

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Fig. 3. Seed germination and seedling growth of 35S-GmWNK1 transgenic seeds. Seeds of wild type, abi4-1, aba2-1 and two lines of transgenic plants were sown on 1/2 MS plates containing different treatments of ABA (0.5 ␮M), NaCl (150 mM) or mannitol (300 mM) for 7 days.

including LEA class genes and ABI1. The expression of LEA genes of RD29A and RD29B (Baker et al., 1994), as well as RAB18 (Lang and Palva, 1992), are known to be induced by ABA and abiotic stresses. In addition, ABI1 encodes a protein phosphatase 2C and regulates ABA signal transduction (Merlot et al., 2001). As shown in Fig. 5, the transcript levels of several ABA-responsive genes were increased in 35S-GmWNK1 transgenic plants with and without ABA conditions(Fig. 5A–D). In contrast, the expression of the coldresponsive genes, COR47 (Thomashow, 1994) and KIN1 (Kurkela and Franck, 1990), increased after ABA treatment, and it differed between genotypes without ABA treatment (Fig. 5E and F). Previously it was demonstrated that GmWNK1 interacted with the ABA catabolic enzyme GmCYP707A1 to fine-tune ABA homeostasis in vivo in soybean (Wang et al., 2010). To test whether overexpression of GmWNK1 affects the Arabidopsis genes encoding homologues of CYP707A (cytochrome P450 monooxygenases), we analyze the expression of CYP707A1-4 genes in wild type and 35S-GmWNK1 transgenic plants under ABA, NaCl and mannitol treatments. Results showed that the expression of AtCYP707A1 and AtCYP707A3 were only detectable under normal conditions, but they were less reduced in response to ABA, NaCl and mannitol in the 35S-GmWNK1 transgenic plants (Fig. 6). In contrast, expression of AtCYP707A2 did not differ significantly between transgenic and wild type plants under both normal and NaCl conditions, but its expression trends are similar to AtCYP707A1/3 under ABA and mannitol conditions (Fig. 6). However, we were not able to detect AtCYP707A4 expression under our present experimental conditions (data not shown). Overall, our results indicated that overexpression of GmWNK1 might also affect the ABA catabolism in Arabidopsis.

Endogenous ABA levels were elevated in 35S-GmWNK1 transgenic plants As described above, 35S-GmWNK1 transgenic plants showed decreased sensitivity to exogenous ABA, NaCl and mannitol, in seed germination rate and seedling development. In addition, the expression levels of ABA responsive genes differed between 35SGmWNK1 transgenic plants and wild type. Moreover, constitutive expression of GmWNK1 also increased endogenous ABA in soybean (Wang et al., 2010). Therefore, the constitutive expression of GmWNK1 in Arabidopsis might have also affected the endogenous levels of ABA. To test this hypothesis, endogenous ABA levels were measured in transgenic plants and wild type plants exposed to normal, NaCl and mannitol conditions. Results showed that it increased in all genotypes, but its level was higher in the transgenic plants (Fig. 7), similar to the data from soybean (Wang et al., 2010), suggesting that the GmWNK1 could be a conserved regulator to ABA homeostasis in plants Discussion In humans, mutations of WNKs cause a Mendelian form of saltsensitive hypertension (Wilson et al., 2001). Several members of this kinase family in humans are salt-sensitive kinases that regulate cell volume homeostasis in response to osmotic stress (Gagnon et al., 2006; Kahle et al., 2006). We previously found that the soybean root-specific protein kinase GmWNK1 interacts with a key ABA 8ˇı-hydroxylase in vitro and in vivo to coordinate ABA homeostasis and its regulation of root development (Wang et al.,

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Fig. 4. Determination of primary root growth of 35S-GmWNK1 transgenic plants. Seeds from wile type and two transgenic lines were sown on plates for 10 days on ABA-free medium and media with ABA (0.5 ␮M), NaCl (150 mM) or mannitol (300 mM). Control refers to the normal condition. WT, wild type. The primary root length was determined using the Image J software; each datum point represents the mean of triplicate experiments (n = 30). **p < 0.01 and *p < 0.05 (t-test); significant difference from the WT under stress condition.

2010). Because mammalian WNKs are responsive to hyperosmotic stresses (Kahle et al., 2008), we have reason to believe that plant WNKs might also play a role in osmotic stresses, which is indeed supported by this study. However, the molecular mechanism of how WNKs act ABA or ABA-dependent under osmotic stress in plants remains unclear. It has been reported that protein phosphorylation events in abscisic acid (ABA) signaling involve several known protein kinases (Finkelstein et al., 2002; Verslues and Zhu, 2007), including the guard cell-specific protein kinase AAPK from Vicia faba and its ortholog OST1/SnRK2.6 in Arabidopsis (Li et al., 2000; Yoshida et al., 2002); SnRKs members of SnRK2.2 and SnRK2.3 (Fujii et al., 2007); receptor for Activated C Kinase 1 (RACK1) (Guo et al., 2009); ABA inducible protein kinase of PKABA1 in barley and wheat (GomezCadenas et al., 2001; Johnson et al., 2008), as well as several calcium dependent protein kinases or the SnRK3/CIPK (Zhu et al., 2007; Cutler et al., 2010). Apart from the above-described kinases, several members of mitogen activated protein kinases (MAPKs) also implicate in ABA signaling (Rodriguez et al., 2010). “MAPK cascade” as a well-established signaling pathway contains MAP3K, MAP2K and MAPK that are activated in series (Rodriguez et al., 2010). However, it is still unclear which upstream components trigger MAPK cascades in plants. In yeast, Ste20p (sterile 20 protein) is a putative mitogen-activated protein kinase kinase kinase

kinase (MAP4K), and its activation triggers downstream three well-defined MAPK cascade, and thereby controls cell growth, and associate with cell-cycle events and osmotic stress (Dan et al., 2001). Ste20 serine/threonine kinase is divided into two groups, the p21-activated kinases (PAKs) and the germinal center kinases (GCKs) (Strange et al., 2006). To date, the STE20/SPS1-related proline/alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1), have been characterized as best WNK substrates, which are activated by WNKs phosphorylation to regulate intracellular ion balance via different co-transporter in response to osmotic stress (Kahle et al., 2008). This evolutionary conserved WNK signaling pathway has been found in C. elegans (WNK-1–GCK-3–CIC anion channel) and its analogs in mammalian (WNK-SPAK–SLC12 co-transporter), indicating that this is a very important network in regulating osmotic homeostasis (Choe and Strange, 2007). Our previous study showed that the Arabidopsis genome has at least ten WNKs, six SPAKs and two OSRs genes (Wang et al., 2008; unpublished data). It is thus plausible to postulate that WNKs involves in ABA signaling via activation of SPAK and OSR1 to trigger MAPK cascade or different co-transporters regulating ABA-dependent osmotic stresses in plants. This ideas is further supported by the observation that mutation of WNK1 exhibited hypersensitivity to ABA (unpublished data), and the single mutant of spaks did not show any defects responding to ABA, while the

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Fig. 5. Expression level analysis of ABA-responsive genes using real time RT-PCR. Expression pattern of ABA-responsive genes, including RD29A, RD29B, RAB18, and ABI1, as well as cold-induced genes COR47 and KIN1 were determined by real-time RT-PCR. Each datum point represents the mean of triplicate replications. **p < 0.01 and *p < 0.05 (t-test); significant difference from the WT under control condition.

double mutants produced various responses to ABA, indicating that they are partially functionally redundant (unpublished data). It would be interesting to investigate whether the existence of the pathway regulates ABA-dependent osmotic stress in plants. As mentioned above, we propose that WNKs participate in ABA signaling pathway, possibly through regulating MAPK cascade. In addition, evidence from our previous study also demonstrates that the soybean WNK1 might affect ABA catabolism by interacting with GmCYP707A1, indicating that the second effect of WNKs on ABA could be metabolism related (Wang et al., 2010). In Arabidopsis, there are 4 homologs of CYP707A (CYP707A 1-4) with major functions in ABA catabolism (Kushiro et al., 2004; Nambara and Marion-Poll, 2005). Mutations of each gene all increased sensitivity to ABA or related stresses (Okamoto et al., 2006; Umezawa et al., 2006). AtCYP707A2 plays a major role during seed germination, whereas AtCYP707A1 and AtCYP707A3 are involved in the regula-

tion of seedling development in response to abiotic stresses (Millar et al., 2006). We found that the expression of these three genes were down-regulated in 35S-GmWNK1 transgenic plants compared to wild type (Fig. 6). Moreover, we also examined the expression of NCED genes for ABA synthesis, and found no differences between transgenic plants and wild type (data not shown). The endogenous ABA level was indeed increased to higher level in seedlings of transgenic plants than that in wild type (Fig. 7), which is consistent with the soybean transgenic results. The increase of ABA level has also been observed in several ABA insensitive mutants, such as abi1-5 (Finkelstein et al., 2002) and atcesa8/irx1 (Chen et al., 2005), but the mechanisms are different. ABI1 and ABI2 might form a feedback loop affecting ABA turnover, ABI3-5 are required for ABA signaling, whereas AtCesA8/IRX1is involved in osmotic stress. As mentioned above, GmWNK1 could be an important factor to mediate ABA homeostasis by negatively regulating CYP707As in

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Fig. 6. Expression level of CYP707A genes were determined by semi-quantitative RT-PCR. Two-week-old plants were grown on 1/2 MS agar plates, and then were subjected to different stresses of ABA (100 ␮M), NaCl (150 mM) and mannitol (300 mM) for another 5 h, respectively. PCR cycles for individual genes were labeled on the right. AtACTIN7 was used as a loading control.

Fig. 7. Measurement of endogenous ABA content in wild type and two 35S-GmWNK1 transgenic plants. Two-week-old plants were grown on agar plates under standard condition, and then were subjected to ABA (100 ␮M), NaCl (150 mM) or mannitol (300 mM) treatments. Samples were harvested for ABA quantification by ELISA after 48 h treatment. FWD (fresh dry weight). Control (under normal condition). Error bars indicate standard errors (n = 3). **p < 0.01 (t-test); significant difference from the WT.

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