Plant Physiol. (1999) 119: 1527-1534

An Arabidopsis GSK3/shaggy-Like Gene That Complements Yeast Salt Stress-Sensitive Mutants Is Induced by NaCl and Abscisic Acid

Hai Lan Piao, Kyeong Tae Pih, Jeong Hwa Lim, Shin Gene Kang, Jing Bo Jin, Sung Hee Kim, and Inhwan Hwang^*

Department of Molecular Biology (H.L.P., J.H.L., S.G.K., J.B.J., S.H.K., I.H.), and Plant Molecular Biology and Biotechnology Research Center (K.T.P., I.H.), Gyeongsang National University, Chinju, 660-701, Korea

	ABSTRACT

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GSK3/shaggy-like genes encode kinases that are involved in a variety of biological processes. By functional complementation of the yeast calcineurin mutant strain DHT22-1a with a NaCl stress-sensitive phenotype, we isolated the Arabidopsis cDNA AtGSK1, which encodes a GSK3/shaggy-like protein kinase. AtGSK1 rescued the yeast calcineurin mutant cells from the effects of high NaCl. Also, the AtGSK1 gene turned on the transcription of the NaCl stress-inducible PMR2A gene in the calcineurin mutant cells under NaCl stress. To further define the role of AtGSK1 in the yeast cells we introduced a deletion mutation at the MCK1 gene, a yeast homolog of GSK3, and examined the phenotype of the mutant. The mck1 mutant exhibited a NaCl stress-sensitive phenotype that was rescued by AtGSK1. Also, constitutive expression of MCK1 complemented the NaCl-sensitive phenotype of the calcineurin mutants. Therefore, these results suggest that Mck1p is involved in the NaCl stress signaling in yeast and that AtGSK1 may functionally replace Mck1p in the NaCl stress response in the calcineurin mutant. To investigate the biological function of AtGSK1 in Arabidopsis we examined the expression of AtGSK1. Northern-blot analysis revealed that the expression is differentially regulated in various tissues with a high level expression in flower tissues. In addition, the AtGSK1 expression was induced by NaCl and exogenously applied ABA but not by KCl. Taken together, these results suggest that AtGSK1 is involved in the osmotic stress response in Arabidopsis.

	INTRODUCTION

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Salt stress, such as that caused by high concentrations of NaCl, causes hyperosmotic stress, imbalance in the cellular ion concentration, and general toxicity that adversely affects plant development. Numerous studies have been conducted to delineate the cellular changes that occur upon exposure to osmotic stress (Walton, 1980; DuPont, 1992; Bohnert et al., 1995; Niu et al., 1995). It has been noted that different plant species employ a variety of different mechanisms to cope with osmotic stress. Responses are especially well understood with regard to physiological and metabolic changes (LaRosa et al., 1987; Binzel et al., 1988; Skriver and Mundy, 1990; Delauney and Verma, 1993; Bohnert et al., 1995; Niu et al., 1995). However, not much has been learned about the mechanism of osmotic stress signaling in plants. In contrast, the signaling mechanism has been studied in detail in yeast. There it has been found that the NaCl stress or high osmotic stress signal is mediated by a MAP kinase pathway (Maeda et al., 1994, 1995). Osmosensor molecules, such as Sln1p and Sho1p, which are located on the plasma membrane, initiate the signaling pathway (Ota and Varshavsky, 1993). The signal then reaches various kinases such as Ssk1p, Pbs2p, and Hog1p (Maeda et al., 1994, 1995; Posas et al., 1996). In addition to the MAP kinase pathway, yeast has another signal transduction pathway that is specific for high NaCl stress. This pathway includes calcineurin, a phosphatase dependent on Ca²⁺, and calmodulin (Nakamura et al., 1993; Mendoza et al., 1994; Wieland et al., 1995). Therefore, it is possible that external high NaCl stress increases intracellular Ca²⁺ concentration, which then causes calmodulin to transmit signals to other, downstream components, such as calcineurin (Matheos et al., 1997). It is also very likely that protein kinases and protein phosphatases are involved in signal transduction in plants. In fact, many genes encoding protein kinases have been shown to be induced under high NaCl conditions and under exogenous ABA treatment (Anderberg and Walker-Simmons, 1992; Urao et al., 1994; Hwang and Goodman, 1995; Jonak et al., 1996; Mizoguchi et al., 1996). It has been suggested that protein kinases may be involved in osmotic stress signal transduction. Recently, the genes at the ABA-insensitive 1 and 2 loci of Arabidopsis have been cloned and shown to encode homologs of phosphatases (Leung et al., 1994, 1997; Meyer et al., 1994). These phosphatases have a Ca²⁺-binding domain, which implies that Ca²⁺ may play a role in the osmotic stress signal transduction in plants. Also, the SOS3 gene, the mutation of which renders Arabidopsis hypersensitive to a high NaCl concentration, encodes a Ca²⁺ sensor homolog, which further confirms the involvement of Ca²⁺ in NaCl stress signaling (Liu and Zhu, 1998). However, in most cases the evidence for the involvement of protein kinases in the NaCl stress or osmotic stress signal pathway is rather circumstantial, and to our knowledge no in vivo substrates for the kinases have been isolated. Also, the in vivo targets of the phosphatases remain to be defined.

Originally, GSK3 was identified as a kinase that phosphorylates glycogen synthase (Embi et al., 1980; Woodgett, 1990), but it has been recently shown that GSK3 is involved in developmental processes such as cell fate determination in Drosophila melanogaster (Ruel et al., 1993), Xenopus (Dominguez et al., 1995), and Dictyostelium (Harwood et al., 1995), and in insulin regulation and transcriptional activation of a variety of proteins in mammalian cells (Nikolakaki et al., 1993; Welsh and Proud, 1993). In yeast there are two GSK3 homologs, Mck1p and Mds1p (Neigeborn and Mitchell, 1991; Puziss et al., 1994). Mutation at the MCK1 gene showed a cold-sensitive phenotype, a temperature-sensitive phenotype, and loss of chromosomes during growth on benomyl. Based on these observations it has been suggested that Mck1p plays an important role in the regulation of kinetochore activity and entry into meiosis. In plants many genes encoding homologs of GSK3 have been identified by virtue of the conservation of the primary structure, i.e. the amino acid sequence, and in many cases it has been suggested that they are involved in developmental processes (Bianchi et al., 1993, 1994; Pay et al., 1993; Dercroocq-Ferrant et al., 1995; Jonak et al., 1995; Dornelas et al., 1998).

In an effort to isolate genes involved in the NaCl stress signal transduction pathway, we screened Arabidopsis cDNA clones for complementation of the yeast strain DHT22-1a, which has deletion mutations in both calcineurin genes. In this paper we report that Arabidopsis cDNA AtGSK1 encodes a protein highly homologous to the glycogen synthase kinase 3 of mammalian cells and to the shaggy gene of D. melanogaster, and that it can rescue the NaCl stress-sensitive phenotype of the yeast calcineurin and mck1 mutants. In addition, we show that the expression of the AtGSK1 gene is induced by NaCl and ABA treatments in Arabidopsis.

The accession number for the nucleotide sequence of the AtGSK1 cDNA reported in this article is AF019927.

	MATERIALS AND METHODS

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Screening of cDNA Clones

Northern- and Southern-Blot Analyses

Complementation of the Yeast Mutation

Assay of the PMR2A Gene Activation

Construction of the mck1 Deletion Mutant

	RESULTS

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Isolation of the AtGSK1 cDNA

Since the original cDNA clone was missing 25 amino acid residues from the N terminus, we introduced into the mutant yeast strain the full-length cDNA under the control of the ADH promoter. The yeast transformant was reexamined on a 1.0 M NaCl plate. As shown in Figure 1, AtGSK1 rescued the NaCl stress-sensitive phenotype of the DHT22-1a cells, which confirmed the original finding. This result suggests that the missing 25 amino acid residues may not affect the ability of AtGSK1 to complement the yeast calcineurin mutant.

View larger version (51K): [in this window] [in a new window]   Figure 1. Complementation of DHT22-1a mutant (mt) cells with AtGSK1. The DHT22-1a (MATa trp1 ade2 ura3 can1-100 cmp::LEU2 cmp2::HIS3) mutant cells were transformed with the full-length AtGSK1 under the ADH1 promoter. The transformed cells were plated on a YPD plate containing 1.0 M NaCl and incubated at 30°C for 4 d. wt, Wild type.

Sequence Analysis of AtGSK1

View larger version (85K): [in this window] [in a new window]   Figure 2. Alignment of the amino acid sequence of AtGSK1 with other GSK3 homologs. The deduced amino acid sequence AtGSK1 is aligned with sequences obtained from public databases. ASK-i, ASK-g, GSK3-rat, shaggy-Dr, and MCK1-S.ce indicate the following: ASK-iota (accession no. 1480078), ASK-gamma (accession no. 1170714), rat GSK3 (accession no. 125374), D. melangaster shaggy (accession no. 125701), and yeast Mck1p, respectively. The gaps were introduced to maximize the alignment. The identical amino acid residues between AtGSK1 and other homologs are shaded.

Genomic Structure of the AtGSK1 Gene

View larger version (34K): [in this window] [in a new window]   Figure 3. Southern-blot analysis of the AtGSK1 gene. Genomic DNA was digested with restriction enzymes and size separated onto a 0.8% agarose gel. The DNA was then transferred onto a nylon membrane and UV cross-linked. Hybridization was carried out with the AtGSK1 cDNA at 65°C overnight. E, H, and S indicate EcoRI, HindIII, and SacI, respectively.

Expression of the AtGSK1 Gene

View larger version (59K): [in this window] [in a new window]   Figure 4. Tissue-specific expression of the AtGSK1 gene. Total RNA (15 µg) isolated from various tissues was size separated onto a 1.2% formaldehyde/agarose gel and transferred onto a nylon membrane. The northern-blot gel was stained with EtBr, and the gel photograph was taken before transfer to examine the loading of the RNA samples. The whole cDNA (Whole) or the specific probe (Specific) of the AtGSK1 cDNA (nucleotide positions 1149-1513) was used as the hybridization probe, respectively. Hybridizations were carried out in a church buffer at 65°C overnight. The blots were washed with 2× SSC once at room temperature and twice with 0.1× SSC, 0.1% SDS at 60°C for 20 min. F, L, R, and S indicate flower, leaf, root, and silique, respectively.

Since the AtGSK1 gene was isolated by rescue of a NaCl stress-sensitive yeast mutant, we examined the possibility of induction of the gene's expression under NaCl stress conditions. To apply NaCl stress to Arabidopsis plants, we treated Arabidopsis seedlings grown for 1 week in liquid culture with 0.15 M NaCl or 100 µM ABA. The Arabidopsis seedlings were harvested at various times, and total RNA was prepared to examine the AtGSK1 transcript level. As shown in Figure 5, the AtGSK1 transcript level accumulated to high levels with time upon NaCl treatment. Also, when the Arabidopsis seedlings were treated with exogenous ABA, the transcript level was increased. However, the pattern of induction was different from that of the NaCl treatment. The transcript level was increased within 30 min, reached the maximum at 1 h, and decreased again at 6 h after the ABA treatment. Next, we addressed whether the induction is specific to NaCl stress. We treated Arabidopsis seedlings with 200 mM KCl in a similar manner and examined the level of AtGSK1 transcripts. As shown in Figure 5, the treatment of Arabidopsis seedlings with KCl did not affect the expression of AtGSK1. The results suggested that the induction of AtGSK1 expression is specific to high NaCl stress.

View larger version (43K): [in this window] [in a new window]   Figure 5. Induction of the AtGSK1 gene expression. A, Arabidopsis seedlings grown in Murashige and Skoog liquid medium were treated with 150 mM NaCl and 100 µM ABA for the indicated times. B, Arabidopsis seedlings were treated with 150 mM and 200 mM KCl for 6 h. Total RNA (15 µg) was analyzed by northern-blot analysis using the AtGSK1-specific probe. To examine the loading of the RNA samples, the northern blot was hybridized with 18S rDNA. Con, No treatment.

AtGSK1 Activates the NaCl Stress Signal Pathway in Yeast

View larger version (16K): [in this window] [in a new window]   Figure 6. Activation of the PMR2A gene in calcineurin mutant cells by AtGSK1. The AtGSK1 cDNA was ligated into an expression vector pJG10. The resulting construct was introduced into DHT22-1a cells, and the cells were screened on SC-Trp plates. The transformed yeast cells were then transformed with a reporter construct, PMR2A:lacZ, and the cells were plated on SC-Ura plates. The double transformants were grown in YPD medium at 30°C overnight, and the overnight culture was inoculated to an A600 of 0.1 into YPD medium. The culture was further incubated until mid-log phase, and NaCl was added to 0.5 M NaCl. Cells were harvested at various time points. Cell extracts were prepared and assayed for -galactosidase activity. Three independent experiments were carried out and similar results were obtained. , , and  indicate the wild type with PMR2A:lacZ, the mutant with pJG10 and PMR2A:lacZ, and the mutant with AtGSK1 and PMR2A:lacZ, respectively.

AtGSK1 Complements mck1 Mutant Cells in Yeast

View larger version (37K): [in this window] [in a new window]   Figure 7. Complementation of the mck1 mutant by the AtGSK1 gene. The AtGSK1 gene was introduced into mck1 mutant cells, and the transformed cells were examined for the complementation of temperature sensitivity and NaCl sensitivity of the mutants. The transformed cells were grown liquid SC-Ura medium at 30°C overnight. The overnight cultures were diluted in YPD medium, plated on YPD, and incubated at 37°C for 3 d to examine temperature sensitivity. To examine NaCl sensitivity the cells were plated in a serial dilution on YPD supplemented with 1.0 M NaCl and incubated at 30°C for 3 d. The numbers on the left side indicate the density of yeast cells.

View larger version (57K): [in this window] [in a new window]   Figure 8. Complementation of the calcineurin mutant by the MCK1 gene. The MCK1 gene was introduced into calcineurin mutant (mt) cells, and the transformed cells were examined for the complementation of NaCl sensitivity of the mutants. The transformed cells were grown in liquid SC-Ura medium at 30°C overnight. The overnight cultures were diluted in YPD medium, plated in a serial dilution on YPD supplemented with 1.0 M NaCl, and incubated at 30°C for 3 d. The numbers on the left side indicate the density of yeast cells. wt, Wild type.

View larger version (32K): [in this window] [in a new window]   Figure 9. Induction of PMR2A:lacZ in the mck1 mutant cells. The PMR2A:lacZ and AtGSK1 genes were introduced into the mck1 mutant cells. The -galactosidase activity was assayed at 1 h, as described in the Figure 6 legend. Three independent assays were carried out and showed nearly identical results.

	DISCUSSION

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To understand a mechanism of signal transduction, it is important to identify the components involved in the pathway; however, it is difficult to isolate such components. In this study we exploited the yeast mutant strain DHT22-1a, which has deletions at both genes of the calcineurin catalytic subunit (Nakamura et al., 1993), thus leaving it sensitive to NaCl stress. The Arabidopsis cDNA AtGSK1, which can rescue this yeast calcineurin mutant from demise under high NaCl conditions, encodes a protein highly homologous to GSK3 found in rat and other GSK3-like proteins in other organisms, including Arabidopsis (Woodgett, 1990; Ruel et al., 1993; Blair, 1994; Dornelas et al., 1998). In Arabidopsis alone, more than five genes encoding proteins highly homologous to GSK3/shaggy have been identified (Jonak et al., 1995; Dornelas et al., 1998). However, in most plants it is not known in which biological processes the encoded proteins function. Also, it has not been previously shown that GSK3 homologs are involved in the NaCl stress or osmotic stress signal transduction pathways. The complementation of the NaCl-sensitive phenotype by AtGSK1 in the calcineurin mutant raised a possibility that AtGSK1 is involved in the NaCl stress signal transduction pathway in plants. This notion was further supported by the fact that AtGSK1 can turn on the transcription of the PMR2A gene in yeast.

In yeast two genes, MCK1 and MDS1, encode homologs of GSK3 (Neigeborn and Michell, 1991; Puziss et al., 1994). It is possible that AtGSK1 may functionally replace Mck1p and Mds1p in the NaCl stress signal transduction in the transformed yeast cells. However, previous studies have shown that these proteins are involved in processes such as meiosis and temperature sensitivity in yeast, but it is not known whether these proteins are also involved in the NaCl stress responses. Therefore, in this study we first addressed whether Mck1p and Mds1p play a role in the NaCl stress signal transduction pathway. The mck1 mutant strain, which has an insertion mutation at the MCK1 gene, exhibited an increased sensitivity to a high NaCl condition in addition to the temperature sensitivity. However, the mds1:His3 mutant did not show any noticeable phenotype. Also, the induction level of the PMR2A gene was greatly reduced in the mck1 mutant cells. When AtGSK1 was introduced into the mck1 mutant, AtGSK1 rescued the NaCl stress sensitivity and partially restored the inducibility of the PMR2A gene in the mutant. Therefore, these results suggest that AtGSK1 can functionally replace Mck1p in the NaCl stress response in yeast. Here we did not address the detailed mechanism of the Mck1p in the NaCl stress signal transduction pathway in yeast. Recently, Matheos et al. (1997) have shown that calcineurin directly interacts with Tcn1p to activate the PMR2A gene in yeast, indicating that no additional components are necessary for the activation. However, calcineurin also plays a role in the activation of PMR2A and the NaCl stress response by a Tcn1p-independent pathway. Therefore, it is possible that Mck1p may be involved in this Tcn1p-independent pathway for the activation of the PMR2A gene. Since expression of the PMR2A gene is regulated by various mechanisms, it is equally possible that Mck1p may activate the expression of the PMR2A gene by other pathways, such as the Hog pathway, or by influencing the carbon metabolism in yeast.

Previously, it has been shown that other homologous genes, such as AtK-1 and MsK, are expressed at high levels in floral tissue (Pay et al., 1993; Jonak et al., 1995). Northern-blot analysis suggests that AtK-1 may be involved in developmental processes, as is the case with homologous genes in animals. However, it is more likely that plant GSK3 homologs are involved in several diverse biological processes, an example being the NaCl stress signal transduction pathway. The data presented here strongly suggest that AtGSK1 is involved in the NaCl stress signal transduction pathway in Arabidopsis. Recently, Pardo et al. (1998) have shown that constitutively activated yeast calcineurin in tobacco plants can substantially increase tolerance to high NaCl stress. Therefore, these results suggest that a calcineurin homolog functions in the NaCl-adaptation process in plants. If this is the case in plants, it is possible that AtGSK1 may be involved in a calcineurin signal transduction pathway in plants. The notion that AtGSK1 may be involved in the NaCl stress signal transduction pathway was further supported by induction of AtGSK1 expression upon NaCl and ABA treatments. However, additional studies are needed to fully elucidate the detailed mechanism by which AtGSK1 plays a role in the NaCl stress response in plants.

	FOOTNOTES

   Received August 12, 1998; accepted December 13, 1998.
¹   This work was supported in part by grants from the Korea Ministry of Science and Technology and from Korea Science and Engineering Foundation to the Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University.

	ABBREVIATIONS

Abbreviations: SC, synthetic complete. Ura, uracil. YPD, yeast peptone dextrose.

	ACKNOWLEDGMENTS

We would like to thank Dr. T. Miyakawa (Hiroshima University, Japan) for the yeast strains DHT22-1a and DHT22-1b, and Dr. G.R. Fink (Massachusetts Institute of Technology, Cambridge) for the reporter construct PMR2A:lacZ.

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