Festuca SAPK 4 Actin 0 125 250 500 A 6 h Stress 24 h Stress 48 h Stress 48 h Stress 0 125 Rice SAPK 4 Actin 6 h StressRice SAPK 4 Actin Rice SAPK 4 Actin 24 h Stress

Background: Plants respond to extracellularly perceived abiotic stresses such as low temperature, drought, and salinity by activation of complex intracellular signaling cascades that regulate acclimatory biochemical and physiological changes. Protein kinases are major signal transduction factors that have a central role in mediating acclimation to environmental changes in eukaryotic organisms. In this study, we characterized the function of the sucrose nonfermenting 1related protein kinase2 (SnRK2) SAPK4 in the salt stress response of rice. Results: Translational fusion of SAPK4 with the green fluorescent protein (GFP) showed subcellular localization in cytoplasm and nucleus. To examine the role of SAPK4 in salt tolerance we generated transgenic rice plants with over-expression of rice SAPK4 under control of the CaMV-35S promoter. Induced expression of SAPK4 resulted in improved germination, growth and development under salt stress both in seedlings and mature plants. In response to salt stress, the SAPK4-overexpressing rice accumulated less Na+ and Cland showed improved photosynthesis. SAPK4-regulated genes with functions in ion homeostasis and oxidative stress response were identified: the vacuolar H+-ATPase, the Na+/H+ antiporter NHX1, the Clchannel OsCLC1 and a catalase. Conclusion: Our results show that SAPK4 regulates ion homeostasis and growth and development under salinity and suggest function of SAPK4 as a regulatory factor in plant salt stress acclimation. Identification of signaling elements involved in stress adaptation in plants presents a powerful approach to identify transcriptional activators of adaptive mechanisms to environmental changes that have the potential to improve tolerance in crop plants. Background Plants respond to abiotic stresses such as cold, drought, and salinity by activation of complex intracellular signaling cascades that regulate biochemical and physiological acclimation. In eukaryotes, protein kinases are key elements involved in signal transduction responsive to metabolism, biotic and abiotic stresses inclusive the major environmental factor salinity. Growth of yeast Published: 28 April 2008 BMC Plant Biology 2008, 8:49 doi:10.1186/1471-2229-8-49 Received: 13 September 2007 Accepted: 28 April 2008 This article is available from: http://www.biomedcentral.com/1471-2229/8/49 © 2008 Diédhiou et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Background
Plants respond to abiotic stresses such as cold, drought, and salinity by activation of complex intracellular signaling cascades that regulate biochemical and physiological acclimation. In eukaryotes, protein kinases are key elements involved in signal transduction responsive to metabolism, biotic and abiotic stresses inclusive the major environmental factor salinity. Growth of yeast mutants deficient in the sucrose non-fermenting 1 (SNF1) serine-threonine protein kinase that is related to the mammalian AMP-activated protein kinase was severely inhibited by NaCl indicating a main function of the kinase in regulating adaptative mechanisms to salt stress [1,2]. In plants, a salt-induced mitogen-activated protein kinase (MAPK) has been, for example, identified from alfalfa with SIMK that is activated by the MAPK kinase SIMKK, and involvement of MAPKs in osmotic stress signaling has been shown in tobacco and A. thaliana [3][4][5]. Stress-inducible members within the plant family of serine-threonine protein kinases have been identified within the calciumdependent protein kinases (CDPKs), the CDPK-related kinases (CRKs), the calmodulin-dependent protein kinases (CaMKs), and the SnRKs that are related to SNF1 from yeast. Members of the SnRK1 subgroup function in regulation of metabolism under environmental stress and have a role in plant development [6][7][8]. Protein kinases of the SnRK2 and SnRK3 type are specific for plants and implication in ABA signaling was shown for several members of these groups [6,[9][10][11]. A. thaliana SnRK3 kinases function in sugar and ABA signaling and in salt stress responses [12][13][14]. SnRK3 SOS2 interacts with the Ca 2+ sensor SOS3 and the plasma membrane Na + /H + antiporter SOS1 involved in regulation of intracellular Na + homeostasis is activated via the SOS pathway [15]. Important knowledge on stress-inducible signaling pathways has been mainly derived from studies on the stresssensitive model plants A. thaliana and rice whereas regulatory signaling elements have been rarely identified in naturally stress tolerant species.
The experiments of this study characterize the protein kinase SAPK4 that was identified in a screen for genes regulated by salt stress in the facultative halotolerant grass Festuca rubra ssp. litoralis (red fescue). Expressional analyses in the salt-sensitive rice line IR29 showed down-regulation of the SAPK4 transcript amounts. Over-expression of the rice SAPK4 in rice conferred increased tolerance to salt stress at the seedling stage and in mature plants. In the transgenic rice, Na + and Claccumulation was reduced indicating involvement of SAPK4 in regulation of ion homeostasis. The results presented in this study indicate that SAPK4 is a determinant of plant salt stress acclimation. Identification of signaling transduction elements that have a role in stress adaptation in naturally stress tolerant plants presents a powerful tool to identify transcriptional regulators of adaptive mechanisms to environmental changes that have the potential to improve tolerance in crop plants.

Differences of salt-dependent expression of SAPK4 in rice and in F. rubra ssp. litoralis
The study started from a comparative analysis of salt stress-induced transcriptional responses in the salt-sensitive rice variety Oryza sativa (ssp. indica) line IR29 and in the salt tolerant grass F. rubra ssp. litoralis. Genes were identified that differentially respond to salinity in both species. F. rubra ssp. litoralis is characterized by substantial salt resistance, tolerates up to 500 mM NaCl and continues growth and development with 250 mM NaCl in hydroponic culture (not shown). In contrast, the rice line IR29 is severely damaged by exposure to salt concentrations of 150 mM NaCl [16,17]. The serine-threonine protein kinase SAPK4 was identified in a subtracted cDNA library from F. rubra ssp. litoralis enriched for salt-responsive genes. This experimental approach allowed to identify an EST-sequence from F. rubra ssp. litoralis that shared 89 and 93% identity on the nucleic acid and amino acid level with SAPK4 from rice (not shown). The present study aimed at a detailed analysis of the role of the kinase in plant salt acclimation. By semiquantitative RT-PCR ( Fig. 1) and Northern-type RNA hybridizations (not shown), expression of SAPK4 was detected in non-stressed control plants of F. rubra and rice. In F. rubra, salt stress of 125 mM reduced and of 250 mM and 500 mM NaCl increased the transcript level of SAPK4. In the salt-sensitive rice line IR29, treatment with 125 mM NaCl for up to 48 h caused a decrease of SAPK4 transcript abundance. Due to lethality, 250 mM and 500 mM NaCl were not applied to rice.
For an analysis of the subcellular localization of the SAPK4 protein, constructs for the expression of the SAPK4 open reading frame cDNA fused to the green fluorescent protein (GFP) reporter gene driven by the 35S-CaMV promoter were generated. Onion epidermis cells were transformed with the translational fusion and fluorescence emission of GFP was monitored under a confocal laser scanning microscope (Fig. 2). In cells incubated for 24 hours in 0.5 × MS nutrient medium, strong GFP signals were detected in the nucleus ( Fig. 2A, B). Cells bombarded with the empty vector as a negative control showed no fluorescence (Fig. 2D) As a positive control, onion epidermal cells were transformed with a translational construct of GFP. In this experiment, GFP showed localization throughout the cell with strongest signals in cytoplasm and nucleus (Fig. 2C). To extent the results obtained from the onion epidermal cells system, A. thaliana mesophyll protoplasts were isolated and were similarly transformed with the SAPK4-GFP transcriptional constructs. After incubation of transformed protoplasts for 24 hours, GFP-derived fluorescence emission was also detected in the cytoplasmic compartment (Fig. 2E).
Effect of salt stress on the transcript abundance of SAPK4 in leaves of F. rubra ssp. litoralis and rice grown under control condi-tions and treated with NaCl for 6 h, 24 h, and 48 h, respectively   Three independent transgenic rice lines designated S1, S4, and S5 were identified by kanamycin resistance and by the presence of the kanamycin resistance gene. Plants of the T2 generations were used for further investigations to examine the role of SAPK4 in plant salt tolerance. In nonstressed plants, a moderate increase in the transcript level of SAPK4 could be detected in the transgenic lines compared to wild-type rice indicating tight regulation of SAPK4 mRNA in rice (Fig. 3). No significant difference between wild-type and transgenic lines was seen in the phenotypes, growth rate, and development up to the age of 12 weeks (not shown). Transgenic plants exposed to elevated NaCl concentrations accumulated more SAPK4 transcript compared with wild-type rice (Fig. 3A) and the lines were analyzed for their salt stress responses. In two independent rice lines that were transformed with the empty plant expression vector the transcript amounts of SAPK4 were not changed in comparison to non-transformed wild-type rice (Fig. 3) and the phenotype of these plants was not changed as well (not shown). These data demonstrate that the observed effects of SAPK4 overexpression in transgenic rice are a result of SAPK4 and not of the empty plant expression vector.

SAPK4 is involved in tolerance to salt stress in rice
Wild-type rice and the transgenic lines were grown in hydroponic culture to the age of 3 weeks under control conditions and subsequently were exposed to 150 mM NaCl. At 7 days of salt treatment the transgenic lines displayed improved salt tolerance ( Fig. 3B, C). Salt-treated wild-type rice showed growth inhibition and developed chlorosis and necrosis (Fig. 3). In contrast, growth of the transgenic lines was rather unaffected and chlorosis was not apparent (Fig. 3B). To test developmental dependence of enhanced tolerance to increased NaCl concentrations by SAPK4, germination assays were performed with wildtype rice and T2 S1 and S4 seedlings. Control and transgenic plants were germinated on control nutrition medium and on medium supplemented with NaCl. Results showed that the germination decreased by approximately 20% in wild-type rice by salt treatment whereas germination was not significantly affected in the transgenic lines (Fig. 4A). In addition, leaf growth was inhibited by the salt stress in wild-type control plants whereas in the transgenic lines the leaf size of seedlings was not significantly changed compared with non-stressed control seedlings (Fig. 4B). Northern-type hybridization of the expression of SAPK4 in leaves of wild-type rice (WT) and the SAPK4 over-expressing rice lines S1 and S4 grown under control conditions and analysis of the SAPK4 transcript levels by RT-PCR in leaves of wild-type rice (WT) and the SAPK4 over-expressing rice lines S1, S4, and S5 exposed to 150 mM NaCl for 48 hours. Analysis of the SAPK4 transcript levels by RT-PCR in leaves of wild-type rice (WT) and the rice lines C1 and C2 that were transformed with the empty plant expression vector and that were exposed to 150 mM NaCl for 48 hours is shown as a control. Transcript levels of actin are shown as a loading control.

SAPK4 regulates ion accumulation in salt-stressed rice
Accumulation of Clwas examined in wild-type rice and in the transgenic lines to test whether SAPK4 affects accumulation of Clunder salt stress. Rice plants were grown to the age of 3 weeks and treated with 150 mM NaCl for 48 hours. Under these stress conditions, the lines S1 and S4 contained only 60% of the Clcontents of wild-type rice (Fig. 5A). Cation homeostasis was addressed by comparing the contents of Na + , K + , and Ca 2+ in wild-type rice and plants of the line S4 that were exposed to the same stress regime as described above. The line S4 accumulated 60% of Na + and 80% K + in comparison to wild-type plants whereas no differences in Ca 2+ accumulation were observed (Fig. 5B). As a physiological reference chlorophyll a fluorescence kinetics were measured and photosynthetic yield calculated. Exposure to 150 mM NaCl for 48 hours resulted in a decreased photosynthetic activity in wild-type rice whereas no significant change occurred in Ion accumulation and photosynthetic quantum yield ΦPSII in SAPK4-over-expressing rice in response to salt stress Control Stress S1 S1

S4 S4
the lines S1 and S4 in comparison with non-stressed control plants of the same lines (Fig. 5C).
The vacuolar ATPase, the vacuolar Na + /H + antiporter NHX1, voltage-gated Clchannels, and catalase are well established targets of salt-dependent regulation (Fig. 6A) and it appeared interesting to study their transcription in wild-type and transgenic rice under salt stress to identify putative target genes regulated by SAPK4. The expression levels of the vacuolar ATPase, the vacuolar Na + /H + antiporter OsNHX1, the Clchannel OsCLC1, and catalase isozyme A were assessed in wild-type and transgenic rice. The transcript amounts of the V-ATPase subunit B and of the catalase increased by treatment with 100 mM NaCl for 48 hours, whereas the transcript amounts of OsNHX1 and OsCLC1 decreased in response to NaCl stress (Fig. 6B). In addition, we were interested in analyzing transcription of the plasma membrane Na + /H + antiporter SOS1 but were not able to detect its expression in both wild-type and transgenic rice.

Discussion
Environmental stresses as drought, cold, and salinity limit the agricultural yield of rice that is one of the most important crops. Experimental approaches such as forward and reverse genetics and transcriptome analyses have been chosen to identify molecular key factors that regulate acclimation of rice to environmental changes. Overexpression of the rice transcription factor OsDREB1A in A. thaliana induced expression of stress-inducible genes and higher tolerance to drought, high-salt, and freezing stresses [18]. Increased salt tolerance of rice was achieved, for example, by transgenic expression of trehalose biosynthetic genes, a Na + /H + antiporter, an aquaporin, the Ca 2+dependent protein kinase OsCDPK7, and the mitogenactivated protein kinase OsMAPK5 [19][20][21][22][23]. A comparison of high-yielding but stress-sensitive rice cultivars with rice varieties with increased stress tolerance indicates that saltsensitive rice varieties are hampered by delayed stressinduced transcriptional response [24].
In the present study a subtractive cDNA library of the halotolerant grass F. rubra ssp. litoralis was screened for transcripts that might be regulated differently by salt stress in F. rubra ssp. litoralis and the salt-sensitive rice line IR29. The identified protein kinase SAPK4 belongs to the rice SnRK2 (sucrose nonfermenting 1-related protein kinase2) family and demonstrated that the kinase mediates salt stress signaling in rice. Constitutive over-expression of rice SAPK4 conferred increased salt tolerance to rice by interfering with ion homeostasis, maintaining unperturbed photosynthesis and inducing an oxidative stress response. Our results demonstrate that the salt-sensitive crop species rice and the related halotolerant grass F. rubra ssp. litoralis differ from each other in the salt-dependent

Control Stress
A B regulation of SAPK4 that apparently plays a role in salt stress signaling. Improved activation of molecular mechanisms of salt adaptation may be seen in transgenic rice plants over-expressing SAPK4. Thus, our results contribute to the understanding of signaling factors that regulate plant salt acclimation. In addition, characterization of SAPK4 and identification of target genes that are regulated either directly or by secondary effects by the kinase extends our knowledge on the function of the rice SnRK2 kinase.
The plant SNF1-related kinases (SnRK) that share homology with the yeast SNF1-type kinases have been divided in the three subgroups SnRK1, SnRK2, and SnRK3 based on domain structure [6,7,25]. SnRK1-type proteins have been reported to function in plant development and carbon metabolism such as pollen development in wheat and regulation of enzymes such as an alpha-amylase in wheat embryos and ADP-glucose pyrophosphorylase in potato tubers [26][27][28]. The wheat SnRK2-subgroup protein PKABA1 is up-regulated by dehydration, cold, and osmotic stress, and involvement in abscisic acid and gibberellin signaling has been shown for the barley homologue [29-32]. Kobayashi et al. [32] analyzed the transcription of SAPK4 in leaf blades, sheaths, and roots of 30-days-old rice under control conditions, ABA, NaCl, and mannitol treatment and found increased transcription in roots and blades by treatment with ABA and NaCl. In this work the authors found regulation of SnRK2 family members by phosphorylation [32]. In other studies, in the rice genome 10 SnRK2s could be identified that were activated by hyperosmotic stress, and 3 of the proteins responded to abscisic acid whereas in A. thaliana 9 of 10 SnRK2s were regulated by hyperosmolarity but not cold indicating function of the kinases in osmotic stress signaling [33,34]. Over-expression of SnRK2.8 improves drought tolerance in A. thaliana but did not regulate stomatal movement whereas SnRK2.6 affects ABA-induced stomatal closure [35]. Members of the A. thaliana SnRK3 group interact with calcium-binding proteins and have a role in sugar and abscisic acid signaling and in salt stress responses [14].
For a more detailed characterization of rice SAPK4 the subcellular partitioning of SAPK4 proteins was addressed by localization of GFP fusions and it was found that the protein kinase was distributed in nucleus and cytoplasm. In yeast it has been shown that the beta subunits of the SNF1 kinase regulate its subcellular localization to the nucleus, vacuole, and cytoplasm [36]. Using GFP protein fusions it was shown that SNF1 kinase beta subunits direct the kinase to the nucleus in a glucose-regulated manner [36]. Direct regulatory interaction between signal transduction pathways mediated by the yeast SNF1 kinase and RNA polymerase II holoenzyme has been suggested to activate transcription of glucose-responsive genes [37]. Accordingly, subcellular localization of rice SAPK4 in both cytoplasm and nucleus may indicate similar regulatory mechanisms of transcriptional control by the kinase in plant cells.
We generated transgenic rice lines over-expressing SAPK4. We found an increased transcript level of SAPK4 in comparison to wild-type rice under non-stress control conditions that was, however, more pronounced under salt treatment. A similar effect has been described for other transcripts as well. Shi et al. [46] reported no increased transcript level of the plasma membrane Na + /H + antiporter SOS1 in SOS1-overexpressing A. thaliana when overexpression was driven by the CaMV-35S promoter. The transcript level increased under treatment with NaCl suggesting postranscriptional regulation of SOS1. The authors suggested that the SOS1 transcript might be unstable in the absence of salt stress and that salt stress causes a stabilization of the SOS1 transcript.
Over-expression of SAPK4 in transgenic rice plants improved germination, growth and development at both the seedling and the mature plant stage in the presence of increased NaCl concentrations whereas wild-type rice showed severe developmental and physiological inhibition under the same conditions. The SAPK4 over-expressing plants accumulated less Na + and Clthan salt-stressed wild-type rice in response to salt stress. The K + /Na + ratio was increased in the SAPK4-sense plants. In parallel photosynthesis was not impaired in the salt-stressed transgenic rice. Identification of target genes indicates that SAPK4 regulates the expression of genes that are known to contribute to ion homeostasis and oxidative stress responses: the vacuolar H + -ATPase, the Na + /H + -antiporter NHX1, the Clchannel OsCLC1, and a catalase.
The vacuolar H + -ATPase mediates electrogenic translocation of protons at endo-membrane compartments of plant cells and energizes processes as cell expansion, secondary activated transport, and adaptation to environmental stress such as salt-induced secondary activated Na + transport via NHX-type Na + /H + antiporters at the tonoplast [38,39]. Stimulated transcription, translation, and enzyme activity, respectively, is known from halophytes as Mesembryanthemum crystallinum and Suaeda salsa and, for example, from the V-ATPase subunit A but not subunit D from A. thaliana [38,[40][41][42]. Over-expression of the vacuolar NHX1-type Na + /H + transporter that mediates vacuolar Na + sequestration improved salt tolerance in tomato and rice [43,44]. SAPK4 over-expressing rice plants, however, revealed reduced transcript amounts of OsNHX1 and a decreased Na + accumulation indicating that the improved tolerance to salt was caused by cellular Na + exclusion rather than vacuolar sequestration of the ion.
For example, suppression of the Na + /K + co-transporter HKT1 reduced Na + accumulation in wheat roots and resulted in increased salt tolerance [45], and reduced accumulation of Na + was induced in A. thaliana by overexpressing the Na + /H + antiporter SOS1 that mediates cellular extrusion of Na + at the plasma membrane [46]. Voltage-dependent Clchannels of the CLC-family function in regulation of membrane potential and cellular pH homeostasis, and involvement of plant CLC-type chloride channels in regulation of stomatal movement has been suggested [47]. In rice, expression of the CLC-type channel OsCLC1 was analyzed showing salt-dependent transcriptional regulation [48]. In the present work transgenic over-expression of SAPK4 in rice repressed both accumulation of Cland transcription of OsCLC1 indicating involvement of the kinase in regulation of anion homeostasis in salt-treated rice. A role of down-regulation of Clchannels as OsCLC1 in the maintenance of turgor and of the intracellular osmotic potential by restricting Clfluxes across the plasma membrane has been hypothesized [48].
In addition to hyperosmotic and hyperionic effects of high salinity, salt-stressed plants are also affected by secondary stresses as excessive generation of reactive oxygen species (ROS). ROS formation is caused by water deficits in salt treated plants that lead to reduced CO 2 fixation and reduced regeneration of NADP + in the Calvin cycle [49]. Reactive oxygen species are scavenged by antioxidant metabolites as ascorbate, glutathione, and tocopherols and by detoxifying enzymes as superoxide dismutase, ascorbate peroxidase, and catalase [50][51][52]. For example, over-expression of glutathione S-transferase and glutathione peroxidase increased growth of transgenic tobacco exposed to salt stress, and transgenic tobacco with reduced catalase activity showed increased susceptibility to salt [53,54]. In this study over-expression of SAPK4 was shown to affect expression of a catalase in rice.

Conclusion
In future investigations it will be interesting to determine the detailed function of this enzyme in the salt acclimation in rice to further advance the understanding of adaptive cellular mechanisms in salt-stressed plants.
Summarizing, the results presented in this study demonstrate that SAPK4 acts as a regulator of salt acclimation in rice that controls ionic homeostasis and photosynthetic activity and allows continued growth and development in the presence of increased salinity (Fig. 7). The experimental data summarized in the model shown in Fig. 7 are Model on the putative involvement of the SNF1-type serine-threonine protein kinase SAPK4 in the regulation of gene expres-sion in response to salinity Figure 7 Model on the putative involvement of the SNF1-type serine-threonine protein kinase SAPK4 in the regulation of gene expression in response to salinity. Catalases are involved in intracellular ROS detoxification and maintenance of photosynthesis [for example 54], the vacuolar ATPase (VHA) energizes the tonoplast NHX-type Na + /H + antiporter for vacuolar Na + sequestration [55], and transcription of voltage gated Cl --channels is regulated salt-dependently in rice [48].

Salt Stress
Cellular Na + and Cl -Export and Sequestration and 150 mM NaCl that is a severe stress for rice [16]. Germination and growth of seedlings were monitored at 50 mM NaCl. For studies of the transcription of genes regulated by SAPK4 the moderate salt stress of 100 mM NaCl was applied.
Construction of subtraction cDNA-library mRNA was isolated from total RNA with the PolyATract kit (Promega, Mannheim, Germany). A subtraction cDNA-library of F. rubra ssp. litoralis was synthesized with the PCR-Select Kit (Clontech, Heidelberg, Germany) according to the manufacturer's protocol. Same amounts of mRNA from the salt stress treatments of F. rubra ssp. litoralis were pooled for the tester cDNA: 125 mM NaCl, 250 mM NaCl, and 500 mM NaCl for 6 h, 24 h, 48 h, and 7 days at the ages of 5 and 12 weeks, each leaf and root tissue. Same amounts of mRNA from control plants of the same developmental stage and harvested in parallel to the stressed plants were pooled for the driver cDNA. The subtracted cDNA was cloned into pCR-TOPO II (Invitrogen, Karlsruhe, Germany) and the inserts were amplified by PCR with the nested primers 1 and 2R (Clontech). The PCR products were analyzed on agarose gels and products that yielded single bands were selected for further procedures. BLAST analyses of sequenced PCR products (MWG Biotech, SeqLab, Germany) were performed in the rice TIGR database.

Preparation of cDNA-macroarrays, labeling of probes, and hybridization of cDNA-arrays
The PCR products from the subtraction cDNA-library were purified with QIAquick spin columns (Qiagen, Hilden, Germany) and dissolved in 50% (v/v) DMSO. cDNA-macroarrays with 129 functionally different ESTs