Identification of Nine Sucrose Nonfermenting 1-related Protein Kinases 2 Activated by Hyperosmotic and Saline Stresses in Arabidopsis thalianaстатья из журнала
Аннотация: Several calcium-independent protein kinases were activated by hyperosmotic and saline stresses in Arabidopsis cell suspension. Similar activation profiles were also observed in seedlings exposed to hyperosmotic stress. One of them was identified to AtMPK6 (Droillard, M. J., Boudsocq, M., Barbier-Brygoo, H., and Laurière, C. (2002) FEBS Lett. 527, 43–50) but the others remained to be identified. They were assumed to belong to the SNF1 (sucrose nonfermenting 1)-related protein kinase 2 (SnRK2) family, which constitutes a plant-specific kinase group. The 10 Arabidopsis SnRK2 were expressed both in cells and seedlings, making the whole SnRK2 family a suitable candidate. Using a family-specific antibody raised against the 10 SnRK2, we demonstrated that these non-MAPK protein kinases activated by hyperosmolarity in cell suspension were SnRK2 proteins. Then, the molecular identification of the involved SnRK2 was investigated by transient expression assays. Nine of the 10 SnRK2 were activated by hyperosmolarity induced by mannitol, as well as NaCl, indicating an important role of the SnRK2 family in osmotic signaling. In contrast, none of the SnRK2 were activated by cold treatment, whereas abscisic acid only activated five of the nine SnRK2. The probable involvement of the different Arabidopsis SnRK2 in several abiotic transduction pathways is discussed. Several calcium-independent protein kinases were activated by hyperosmotic and saline stresses in Arabidopsis cell suspension. Similar activation profiles were also observed in seedlings exposed to hyperosmotic stress. One of them was identified to AtMPK6 (Droillard, M. J., Boudsocq, M., Barbier-Brygoo, H., and Laurière, C. (2002) FEBS Lett. 527, 43–50) but the others remained to be identified. They were assumed to belong to the SNF1 (sucrose nonfermenting 1)-related protein kinase 2 (SnRK2) family, which constitutes a plant-specific kinase group. The 10 Arabidopsis SnRK2 were expressed both in cells and seedlings, making the whole SnRK2 family a suitable candidate. Using a family-specific antibody raised against the 10 SnRK2, we demonstrated that these non-MAPK protein kinases activated by hyperosmolarity in cell suspension were SnRK2 proteins. Then, the molecular identification of the involved SnRK2 was investigated by transient expression assays. Nine of the 10 SnRK2 were activated by hyperosmolarity induced by mannitol, as well as NaCl, indicating an important role of the SnRK2 family in osmotic signaling. In contrast, none of the SnRK2 were activated by cold treatment, whereas abscisic acid only activated five of the nine SnRK2. The probable involvement of the different Arabidopsis SnRK2 in several abiotic transduction pathways is discussed. Environmental stresses such as drought, cold, and salinity impose osmotic stress on plants, leading to imbalance in ionic homeostasis, oxidative damages, and growth inhibition. Understanding how plants respond to these stresses is critical to improve plant resistance. Reversible protein phosphorylation is one of the major mechanisms for mediating intracellular responses, including responses to osmotic changes. Indeed, several protein kinases have been shown to be activated by hyperosmotic stresses in different plant species. Because of the well known osmosensing pathway in yeast involving a mitogen-activated protein kinase (MAPK), 1The abbreviations used are: MAPK, mitogen-activated protein kinase; SnRK2, sucrose nonfermenting 1-related protein kinase 2; SnRK3, sucrose nonfermenting 1-related protein kinase 3; CPDK, calcium-dependent protein kinase; ABA, abscisic acid; MES, 4-morpholinoethanesulfonic acid; HA, hemagglutinin; RT, reverse transcription; DTT, dithiothreitol; MBP, myelin basic protein. much interest was focused on the MAPK family in plants. In Arabidopsis, AtMPK6 and AtMPK4 were shown to be activated by hyperosmolarity, salt, cold, or drought (1Droillard M.J. Boudsocq M. Barbier-Brygoo H. Laurière C. FEBS Lett. 2002; 527: 43-50Crossref PubMed Scopus (146) Google Scholar, 2Ichimura K. Mizoguchi T. Yoshida R. Yuasa T. Shinozaki K. Plant J. 2000; 24: 655-665Crossref PubMed Google Scholar), whereas the tobacco SIPK was activated by hyperosmotic or salt stresses (3Droillard M.J. Thibivilliers S. Cazalé A.C. Barbier-Brygoo H. Laurière C. FEBS Lett. 2000; 474: 217-222Crossref PubMed Scopus (59) Google Scholar, 4Mikolajczyk M. Awotunde O.S. Muszynska G. Klessig D.F. Dobrowolska G. Plant Cell. 2000; 12: 165-178Crossref PubMed Scopus (261) Google Scholar, 5Hoyos M.E. Zhang S.Q. Plant Physiol. 2000; 122: 1355-1363Crossref PubMed Scopus (107) Google Scholar). In alfalfa, SAMK was reported to be activated by cold and drought but not NaCl (6Jonak C. Kiegerl S. Ligterink W. Barker P.J. Huskisson N. Hirt H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11274-11279Crossref PubMed Scopus (395) Google Scholar), whereas SIMK was activated by sorbitol, KCl, and NaCl (7Munnik T. Ligterink W. Meskiene I. Calderini O. Beyerly J. Musgrave A. Hirt H. Plant J. 1999; 20: 381-388Crossref PubMed Google Scholar). In mammals, MAPK cascades are composed of MAPK, MAPKK, and MAPKKK, each component being activated by phosphorylation by the upstream kinase. The involvement of MAPKK and MAPKKK in plant osmotic response was suggested by molecular and biochemical studies. The Arabidopsis MAPKKK AtMEKK1 was transcriptionally induced by salt stress and the protein was able to complement a yeast mutant affected in osmotic signaling (8Covic L. Silva N.F. Lew R.R. Biochim. Biophys. Acta. 1999; 1451: 242-254Crossref PubMed Scopus (26) Google Scholar). Moreover, Mizoguchi et al. (9Mizoguchi T. Ichimura K. Irie K. Morris P. Giraudat J. Matsumoto K. Shinozaki K. FEBS Lett. 1998; 437: 56-60Crossref PubMed Scopus (103) Google Scholar) described using two-hybrid and yeast complementation a possible MAPK cascade composed of the osmotically activated AtMPK4, AtMEK1 (MAPKK), and AtMEKK1. More recently, the activation of AtMPK4 by AtMEK1 (10Huang Y.F. Li H. Gupta R. Morris P.C. Luan S. Kieber J.J. Plant Physiol. 2000; 122: 1301-1310Crossref PubMed Scopus (128) Google Scholar) and the interaction between SIPKK and SIPK (11Liu Y. Zhang S. Klessig D.F. Mol. Plant Microbe Interact. 2000; 13: 118-124Crossref PubMed Scopus (47) Google Scholar) were demonstrated in vitro independently of osmotic signaling. Interestingly, Kiegerl et al. (12Kiegerl S. Cardinale F. Siligan C. Gross A. Baudouin E. Liwosz A. Eklof S. Till S. Bogre L. Hirt H. Meskiene I. Plant Cell. 2000; 12: 2247-2258Crossref PubMed Scopus (195) Google Scholar) demonstrated that SIMKK activated SIMK in vivo and enhanced the SIMK activation by NaCl. Other kinase families have been shown to play a role in osmotic signaling. Among them, SOS2 (salt overly sensitive 2), which belongs to the sucrose nonfermenting 1-related protein kinase 3 (SnRK3) family, was transcriptionally induced by salt stress (13Liu J.P. Ishitani M. Halfter U. Kim C.S. Zhu J.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3730-3734Crossref PubMed Scopus (642) Google Scholar). sos2 mutant displayed hypersensitivity to Na+ ions but not to mannitol (14Zhu J.K. Liu J. Xiong L. Plant Cell. 1998; 10: 1181-1191Crossref PubMed Scopus (555) Google Scholar), suggesting a role of SOS2 in ion homeostasis. Using in vitro and yeast experiments, progress has been made in understanding the SOS2 pathway. Intramolecular interaction maintains SOS2 in an inactive form by autoinhibition (15Guo Y. Halfter U. Ishitani M. Zhu J.K. Plant Cell. 2001; 13: 1383-1399Crossref PubMed Scopus (399) Google Scholar), which is relieved by interaction with the calcium-binding protein SOS3 (16Ishitani M. Liu J. Halfter U. Kim C.S. Shi W. Zhu J.K. Plant Cell. 2000; 12: 1667-1678Crossref PubMed Scopus (396) Google Scholar, 17Halfter U. Ishitani M. Zhu J.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3735-3740Crossref PubMed Scopus (620) Google Scholar). Then SOS3 targets SOS2 to the plasma membrane where the SOS2-SOS3 complex activates the SOS1 Na+/H+ antiporter via phosphorylation (18Quintero F.J. Ohta M. Shi H.Z. Zhu J.K. Pardo J.M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9061-9066Crossref PubMed Scopus (448) Google Scholar, 19Qiu Q.S. Guo Y. Dietrich M.A. Schumaker K.S. Zhu J.K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8436-8441Crossref PubMed Scopus (916) Google Scholar). Moreover, the complex also induces the transcriptional up-regulation of SOS1 by salt stress (20Shi H. Ishitani M. Kim C. Zhu J.K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6896-6901Crossref PubMed Scopus (1260) Google Scholar). Calcium signals elicited by hyperosmotic stresses (21Sanders D. Pelloux J. Brownlee C. Harper J.F. Plant Cell. 2002; 14: S401-S417Crossref PubMed Scopus (1001) Google Scholar) can also be sensed by calcium-dependent protein kinases (CDPK), although very few data are available on the role of CDPK in osmotic signaling. Transcriptional induction of several CDPK was reported in response to salt, cold, or drought in different plant species (22Urao T. Katagiri T. Mizoguchi T. Yamaguchi-Shinozaki K. Hayashida N. Shinozaki K. Mol. Gen. Genet. 1994; 244: 331-340Crossref PubMed Scopus (251) Google Scholar, 23Yoon G.M. Cho H.S. Ha H.J. Liu J.R. Lee H.S. Plant. Mol. Biol. 1999; 39: 991-1001Crossref PubMed Scopus (136) Google Scholar, 24Saijo Y. Hata S. Kyozuka J. Shimamoto K. Izui K. Plant J. 2000; 23: 319-327Crossref PubMed Google Scholar). Moreover, overexpression of OsCDPK7 conferred cold, salt, and drought tolerance on rice plants (24Saijo Y. Hata S. Kyozuka J. Shimamoto K. Izui K. Plant J. 2000; 23: 319-327Crossref PubMed Google Scholar). Interestingly, the activation of a rice CDPK in response to cold treatment was reported, but it occurred only 12 to 18 h after treatment, indicating that the kinase does not participate in the early response to stress but rather in the adaptative process (25Martin M.L. Busconi L. Plant Physiol. 2001; 125: 1442-1449Crossref PubMed Scopus (104) Google Scholar). Transcript accumulation in response to NaCl or PEG treatments was also reported for several SHAGGY/GSK3-like kinases (AtSK) (26Piao H.L. Pih K.T. Lim J.H. Kang S.G. Jin J.B. Kim S.H. Hwang I. Plant Physiol. 1999; 119: 1527-1534Crossref PubMed Scopus (83) Google Scholar, 27Charrier B. Champion A. Henry Y. Kreis M. Plant Physiol. 2002; 130: 577-590Crossref PubMed Scopus (160) Google Scholar). On the other hand, AtSK22 complemented a yeast salt stress-sensitive mutant (26Piao H.L. Pih K.T. Lim J.H. Kang S.G. Jin J.B. Kim S.H. Hwang I. Plant Physiol. 1999; 119: 1527-1534Crossref PubMed Scopus (83) Google Scholar) and its overexpression enhanced salt and drought tolerance in Arabidopsis (28Piao H.L. Lim J.H. Kim S.J. Cheong G.W. Hwang I. Plant J. 2001; 27: 305-314Crossref PubMed Google Scholar). Only few data are available on another group of the SNF1-related protein kinases (SnRK2), which appears unique to plants (29Hrabak E.M. Chan C.W. Gribskov M. Harper J.F. Choi J.H. Halford N. Kudla J. Luan S. Nimmo H.G. Sussman M.R. Thomas M. Walker Simmons K. Zhu J.K. Harmon A.C. Plant Physiol. 2003; 132: 666-680Crossref PubMed Scopus (786) Google Scholar). A tobacco homolog of the Arabidopsis ASK1/SnRK2–4 was shown to be activated by hyperosmotic stress in cell suspension (4Mikolajczyk M. Awotunde O.S. Muszynska G. Klessig D.F. Dobrowolska G. Plant Cell. 2000; 12: 165-178Crossref PubMed Scopus (261) Google Scholar), whereas dehydration activated SnRK2-E/SnRK2–6 (30Yoshida R. Hobo T. Ichimura K. Mizoguchi T. Takahashi F. Aronso J. Ecker J.R. Shinozaki K. Plant Cell Physiol. 2002; 43: 1473-1483Crossref PubMed Scopus (442) Google Scholar). In soybean, SPK3 and SPK4 were transcriptionally induced by drought and saline stress (31Yoon H.W. Kim M.C. Shin P.G. Kim J.S. Kim C.Y. Lee S.Y. Hwang I. Bahk J.D. Hong J.C. Han C. Cho M.J. Mol. Gen. Genet. 1997; 255: 359-371Crossref PubMed Scopus (33) Google Scholar), whereas only SPK1 and SPK2 were activated by salt stress in yeast (32Monks D.E. Aghoram K. Courtney P.D. DeWald D.B. Dewey R.E. Plant Cell. 2001; 13: 1205-1219Crossref PubMed Scopus (102) Google Scholar). We have previously reported the activation of several protein kinases that did not require calcium for their activity, in response to hyperosmotic stresses in Arabidopsis cell suspension (1Droillard M.J. Boudsocq M. Barbier-Brygoo H. Laurière C. FEBS Lett. 2002; 527: 43-50Crossref PubMed Scopus (146) Google Scholar). Among them, one was identified to the MAPK AtMPK6 but the others remained unknown. In this work, we demonstrated that these unidentified kinases were also activated in Arabidopsis seedlings and belonged to the SnRK2 family. Molecular identification revealed that nine of the 10 SnRK2 were activated by hyperosmolarity. Then the possible involvement of SnRK2 in other signal transduction pathways such as saline stress, cold, and abscisic acid (ABA) was also tested. Plant Material—Arabidopsis thaliana cell suspension (Columbia ecotype) was cultured in JPL medium as previously described (1Droillard M.J. Boudsocq M. Barbier-Brygoo H. Laurière C. FEBS Lett. 2002; 527: 43-50Crossref PubMed Scopus (146) Google Scholar) at 23 °C in constant light. Cells were used after 5 days subculturing with 100 mg of fresh weight/ml cell density. A. thaliana (Columbia ecotype) seeds were sterilized and sown on a medium containing 5 mm KNO3, 2.5 mm K2HPO4/KH2PO4, pH 6, 2 mm MgSO4, 1 mm Ca(NO3)2, 1 mm MES, 50 μm Fe-EDTA, Murashige and Skoog microelements (33Murashige T. Skoog F. Physiol. Plant. 1962; 15: 473-497Crossref Scopus (54341) Google Scholar), 10 g/liter sucrose, and 7 g/liter agar. The seedlings were grown at 21 °C with a 8-h dark/16-h light (110 μE m–2 s–1) photoperiod. Expression and Purification of His-tagged SnRK2—PCR was performed using the primers presented in Table I. The open reading frame of SnRK2-1, SnRK2-2, SnRK2-3, SnRK2-4, SnRK2-5, SnRK2-7, and SnRK2-10 were cloned as a KpnI-PstI fragment into pQE30 (Qiagen) and the open reading frame of SnRK2–6, SnRK2–8, and SnRK2–9 as a SphI-KpnI fragment. After transformation of Escherichia coli DH5α, positive clones containing the fragments were verified by DNA sequencing. His-tagged proteins were expressed in M15 E. coli (Qiagen) and purified using nickel columns according to the manufacturer's instructions (Qiagen).Table IOligonucleotides used for SnRK2 cloning into pQE30 and RT-PCRGeneSequence of oligonucleotides forward (F) and reverse (R)SnRK2-1F5′-TTTGGTACCATGGACAAGTATGACGTTG-3′R5′-ATTCTGCAGTTTAAGCTTTGTCAGACTCTTG-3′SnRK2-2F5′-AAAGGTACCATGGATCCGGCGACT-3′R5′-TCCCTGCAGTCAGAGAGCATAAACTATCT-3′SnRK2-3F5′-TTAGGTACCATGGATCGAGCTCCG-3′R5′-GGCTGCAGATTAGAGAGCGTAAACTATC-3′SnRK2-4F5′-TTAGGTACCGCGAGAATGGACAA-3′R5′-CAGACTGCAGACCAAAATATCAACTTATTCT-3′SnRK2-5F5′-TTAGGTACCATGGACAAGTATGAGGTTG-3′R5′-TTTCTGCAGTTAAGCTTTGGGAGGC-3′SnRK2-6F5′-TTAGCATGCATGGATCGACCAGCAG-3′R5′-TTTGGTACCGTATCACATTGCGTACACA-3′SnRK2-7F5′-TTAGGTACCATGGAGAGATACGACATC-3′R5′-TTTCTGCAGTCATAGAGCACATACGAA-3′SnRK2-8F5′-TTAGCATGCATGGAGAGGTACGAAATA-3′R5′-TATGGTACCTTCACAAAGGGGAAAG-3′SnRK2-9F5′-TTAGCATGCATGGAGAAGTATGAGATGG-3′R5′-TTTGGTACCCTATGCGTAATCATCATAC-3′SnRK2-10F5′-TTAGGTACCATGGACAAGTACGAGCTT-3′R5′-TTTCTGCAGTTTTAACTGACTCGGACT-3′ Open table in a new tab GATEWAY Cloning of SnRK2—The open reading frame of SnRK2 genes were amplified by PCR using primers containing attB1 and attB2 sequences that were purchased from Invitrogen. The sequences of the oligonucleotides are presented in Table II. For the reverse primers, the stop codon was removed to allow C-terminal fusions. In vitro BP clonase recombination reactions using pDONR207 as entry vector were carried out according to the manufacturer's instructions (Invitrogen). The product of recombination reactions (BP reactions) was used to transform E. coli DH5α, and the positive clones were verified by DNA sequencing.Table IIOligonucleotides used for SnRK2 cloning into pDONR207GeneSequence of oligonucleotides forward (F) and reverse (R)SnRK2-1F5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGACAAGTATGACGTT-3′R5′-GGGGACCACTTTGTACAAGAAAGCTGGGTGAGCTTTGTCAGACTCTTGA-3′SnRK2-2F5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGATCCGGCGACT-3′R5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCGAGAGCATAAACTATCTCTCCA-3′SnRK2-3F5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGATCGAGCTCCG-3′R5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCGAGAGCGTAAACTATCTCT-3′SnRK2-4F5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGACAAGTACGAGCTG-3′R5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCACTTATTCTCACTTCTCCAC-3′SnRK2-5F5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGACAAGTATGAGGTT-3′R5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCAGCTTTGGGAGGCTCT-3′SnRK2-6F5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGATCGACCAGCAG-3′R5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCCATTGCGTACACAATCTC-3′SnRK2-7F5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGAGAGATACGACATC-3′R5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTAGAGCACATACGAAATC-3′SnRK2-8F5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGAGAGGTACGAAATA-3′R5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCCAAAGGGGAAAGGAGA-3′SnRK2-9F5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGAGAAGTATGAGATGG-3′R5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCTGCGTAATCATCATACCA-3′SnRK2-10F5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCACCATGGACAAGTACGAGCTT-3′R5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCACTGACTCGGACTTCTC-3′ Open table in a new tab LR clonase recombination reactions to transfer DNA fragments from entry clones to destination vectors were carried out according to the manufacturer's instructions (Invitrogen) using pGreen-HiA-GW (see below) as destination vector. The product of recombination reactions (LR reactions) was used to transform E. coli DH5α. Construction of Destination Vector pGreen-HiA-GW—The bluntended GATEWAY conversion cassette C (Invitrogen) was inserted in the BamHI site of the intron-tagged HA-epitope cassette of pPILY vector (34Ferrando A. Farras R. Jasik J. Schell J. Koncz C. Plant J. 2000; 22: 553-560Crossref PubMed Google Scholar) after filling in the site with Klenow polymerase I. The resulting cassette was inserted in the KpnI site of pGreen0129 (www.pGreen.ac.uk). This construction allows the expression of HA-tagged proteins under the control of a 35 S promoter carrying a duplicated enhancer domain. RT-PCR Analysis—Total RNA was isolated from cell suspension 5 days after subculturing and from 6-day-old seedlings using NucleoSpin RNA Plant kit (Macherey-Nagel). The cDNA was synthesized from 4 μg of total RNA and oligo(dT) using the Superscript First-Strand Synthesis System (Invitrogen). PCR was run for 30–40 cycles using the primers presented in Table I. Amplified transcripts were separated on 0.8% (w/v) agarose gel and detected by ethidium bromide staining. The gene specificity of the primers was confirmed by subsequent cloning and sequencing of the amplified products (see section “Expression and Purification of His-tagged SnRK2”). As an internal standard, the level of ACTIN2 and ACTIN8 transcripts was monitored with the following primers: 5′-GATTCAGATGCCCAGAAGTCTTG-3′ (forward) and 5′-GCTGGAATGTGCTGAGGGAAG-3′ (reverse), because their combined expression profile was shown to be constitutive (35An Y.Q. McDowell J.M. Huang S. McKinney E.C. Chambliss S. Meagher R.B. Plant J. 1996; 10: 107-121Crossref PubMed Scopus (408) Google Scholar). Cell Suspension Treatments—Osmolarity was monitored using a freezing-point osmometer (Roebling, Berlin, Germany). Cells were equilibrated for 4 h in their culture medium containing 10 mm MES-Tris, pH 6.2, and adjusted to 200 mOsm with sucrose. After equilibration, extracellular medium was replaced by either the same volume of isoosmotic medium, 200 mOsm (10 mm MES-Tris, pH 6.2, 1 mm CaSO4, 190 mm sucrose), or hyperosmotic medium, 500 or 1000 mOsm (10 mm MES-Tris, pH 6.2, 1 mm CaSO4, 500 or 1000 mm sucrose). In the indicated cases, sucrose was replaced by 1000 mm mannitol or 650 mm NaCl to get similar hyperosmolarity. To stop treatment, cell suspension was filtered, frozen in liquid nitrogen, and stored at –80 °C until use. Seedling Treatments—Seedlings were grown for 6 days in the medium described above (control medium). Plantlets were transferred to either isoosmotic medium (control medium) or to the same medium containing 500 or 1000 mm sucrose for 30 min. To stop treatment, seedlings were frozen in liquid nitrogen and stored at –80 °C until use. Preparation of Protoplasts—Cell cultures were used 3 days after subculturing at 33% (v/v) for isolation of protoplasts. Cells were collected by centrifugation (540 × g for 5 min) and resuspended in 25 ml of enzymatic solution (1% (w/v) cellulase RS (Yakult, Tokyo, Japan), 0.2% (w/v) macerozyme R10 (Yakult) in modified JPL medium (JPL-A), which does not contain sucrose but 30.5 g/liter glucose and 30.5 g/liter mannitol). Final volume was adjusted to 75 ml with JPL-A and cells were incubated for 3 h 30 min on a rotary shaker in constant light. Protoplasts were collected by centrifugation (150 × g for 5 min), washed with JPL-A, and resuspended in JPL medium containing 0.28 m sucrose. After centrifugation (150 × g for 5 min), floating protoplasts were harvested. Protoplast Transient Expression Assay—Typically, 1.5 × 106 protoplasts in 120 μl were mixed with 20–40 μg of plasmid DNA and 360 μl of PEG solution (25% (w/v) PEG 6000, 450 mm mannitol, 100 mm Ca(NO3)2). After an incubation at 22 °C in the dark for 15 min, the transfection mixture was washed with 275 mm Ca(NO3)2 and centrifuged at 150 × g for 5 min. Protoplasts were resuspended in 1 ml of JPL-A and incubated in the dark at 22 °C for 15 h before treatments. Protoplast Treatments—For osmotic stresses, protoplasts were centrifuged (150 × g for 5 min) and resuspended in the same volume of isoosmotic medium (JPL-A, 400 mOsm) or hyperosmotic medium containing mannitol (JPL-A supplemented with 660 mm mannitol, 1000 mOsm) or NaCl (JPL-A supplemented with 350 mm NaCl, 1000 mOsm) for 10 min. For cold treatment, protoplasts were incubated for 10 min at 4 °C or room temperature for the control. For ABA treatment, protoplasts were incubated for 20 min with 30 μm ABA or the same volume of ethanol solvent control. To stop treatments, protoplasts were centrifuged (150 × g for 5 min) and then the pellet was frozen in liquid nitrogen before storage at –80 °C until use. Preparation of Protein Extracts—Cells or seedlings were ground in liquid nitrogen and homogenized at 4 °C in extraction buffer EB1 (100 mm HEPES, pH 7.5, 5 mm EDTA, 5 mm EGTA, 2 mm orthovanadate, 10 mm NaF, 20 mm β-glycerophosphate, 5 mm DTT, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml antipain). After centrifugation at 17,600 × g at 4 °C for 15 min, the supernatant was precipitated in 10% (w/v) trichloroacetic acid solution containing 10 mm NaF, washed twice with 80% (v/v) cold acetone, and resuspended in SDS-PAGE sample buffer. Protein concentration was determined by the Bradford method (36Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217548) Google Scholar). For immunoprecipitation, the 17,600 × g supernatant was obtained as above in extraction buffer EB2 (EB1 modified in the concentration of three protectants: DTT (10 mm), orthovanadate (10 mm), and β-glycerophosphate (60 mm)). For protein extraction from protoplasts, extraction buffer EB3 corresponds to EB2 supplemented with 1% (v/v) Triton X-100 and 75 mm NaCl. The protoplast pellet was melted on ice with 100 μl of EB3 and vortexed. Protein extract was recovered after centrifugation at 17,600 × g at 4 °C, frozen in liquid nitrogen, and stored at –80 °C until use. In-Gel Kinase Assay—Protein extract (20 μg) was separated on 10% SDS-polyacrylamide gels embedded with 0.2 mg/ml myelin basic protein (MBP) or 0.5 mg/ml histone as substrates for the kinases. The gels were treated for protein renaturation as described by Zhang et al. (37Zhang S. Du H. Klessig D.F. Plant Cell. 1998; 10: 435-449PubMed Google Scholar). For the activity, the gels were preincubated for 30 min at room temperature in kinase activity buffer (40 mm HEPES, pH 7.5, 2 mm DTT, 20 mm MgCl2, 1 mm EGTA, 0.1 mm orthovanadate). Phosphorylation was performed for 1 h in 8 ml of the same buffer supplemented with 25 μm cold ATP and 2.9 MBq of [γ-33P]ATP per gel. Then the gels were washed extensively in 5% (w/v) trichloroacetic acid and 1% (w/v) disodium pyrophosphate solution. The protein kinase activity was detected on the dried gels by the Storm imaging system (Amersham Biosciences). Immunoprecipitation—Immunoprecipitation assays were carried out either with the polyclonal anti-HA antibody (Sigma) or with a polyclonal plant kinase antibody. It was raised against 15 amino acids of the catalytic domain of SnRK2 kinases (GYSKSSLLHSRPKST, Eurogentec). Protein extract (200 μg from cells or 100 μg from protoplasts) was incubated with either 35 μg of anti-SnRK2 or 1.6 μg of anti-HA (Sigma) antibodies in immunoprecipitation buffer (20 mm HEPES, pH 7.5, 5 mm EDTA, 5 mm EGTA, 0.1 mm orthovanadate, 10 mm NaF, 60 mm β-glycerophosphate, 5 mm DTT, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 5 μg/ml antipain, 150 mm NaCl, 0.5% (v/v) Triton X-100, 0.5% (v/v) Nonidet P-40) for 3 h. Then 30 μl of 50% protein A-Sepharose CL-4B (Sigma) was added and incubation was continued for another 1 h. The immunoprecipitate was washed 4 times in immunoprecipitation buffer and twice in kinase buffer (20 mm Tris-HCl, pH 7.5, 12 mm MgCl2, 2 mm EGTA, 2 mm DTT, 0.1 mm orthovanadate) and resuspended in SDS-PAGE sample buffer. Kinase activity of precipitated proteins was analyzed by the in-gel kinase assay as previously described. Competition was performed with two different peptides: the SnRK2 peptide used for immunization and one AtSK peptide (KKVLQDRRYKNRELQC, Eurogentec). The antibody was preincubated for 10 min with the peptide before adding protein extract and immunoprecipitation buffer. The peptide was used at a final concentration of 0.2 mm. Immunoblotting—Recombinant proteins (100 ng) or total extracts (10 μg from protoplasts or 20 μg from cells) were separated on 10% SDS-polyacrylamide gels and immunoblotted onto polyvinylidene difluoride membranes (Millipore). Blots were blocked with 5% (w/v) defatted milk in TBS-Tween (10 mm Tris-HCl, pH 7.5, 154 mm NaCl, 0.1% (v/v) Tween 20) and probed with either 1:2000 polyclonal anti-SnRK2 or 1:5000 monoclonal anti-HA (Sigma) antibodies. Alkaline phosphatase-conjugated anti-rabbit IgG (Bio-Rad) or horseradish peroxidase-conjugated anti-mouse IgG (Sigma) were used as secondary antibodies and the reactions were visualized using the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate kit (Bio-Rad) and enhanced chemiluminescence ECL kit (Amersham Biosciences), respectively. Accession Numbers—The GenBank™ accession numbers for the sequences described in this article are as follows: NM_120946 (SnRK2-1), NM_114910 (SnRK2-2), NM_126087 (SnRK2-3), NM_100969 (SnRK2-4), NM_125760 (SnRK2-5), NM_119556 (SnRK2-6), NM_120165 (SnRK2-7), NM_106478 (SnRK2-8), NM_127867 (SnRK2-9), NM_104774 (SnRK2-10), BAB61735 (PKABA1), AF186020 (AAPK), U41998 (ACTIN2), and U42007 (ACTIN8). Several Non-MAPK Protein Kinases Are Activated by Hyperosmotic and Saline Stresses in Arabidopsis Cells and Plantlets—To investigate the activation of Ca2+-independent protein kinases in response to hyperosmotic stresses, in-gel kinase assays were performed in the absence of calcium using MBP or histone as substrates. Moderate (500 mOsm) or high (1000 mOsm) hyperosmotic stresses were studied in comparison to an isoosmotic control condition as previously described (1Droillard M.J. Boudsocq M. Barbier-Brygoo H. Laurière C. FEBS Lett. 2002; 527: 43-50Crossref PubMed Scopus (146) Google Scholar). When Arabidopsis cell suspension was submitted to hyperosmolarity, the activation of several Ca2+-independent kinases was observed using MBP as a substrate: two thin activity bands with apparent molecular masses of 42 and 35 kDa and a thick one around 37–38 kDa (Fig. 1A). The activations were greater when the stress strength was increased. When the same extracts were analyzed using histone as a substrate, a similar activation profile was visualized, with two distinct bands with apparent molecular masses of 38 and 37 kDa in addition to the 42- and 35-kDa bands. These four protein kinases activated by hyperosmolarity can phosphorylate both histone and MBP, indicating that they do not belong to the MAPK family. By contrast, the 44-kDa kinase active on MBP but almost not on histone has already been identified to the MAPK AtMPK6 (1Droillard M.J. Boudsocq M. Barbier-Brygoo H. Laurière C. FEBS Lett. 2002; 527: 43-50Crossref PubMed Scopus (146) Google Scholar). The effect of two other osmolytes, mannitol and NaCl, was analyzed in conditions leading to the same osmolarity (Fig. 1B). When cells were treated with 1 m sucrose, 1 m mannitol, or 650 mm NaCl, similar activation profiles were observed, indicating that these kinase activations represent a general response to osmotic stress. When Arabidopsis seedlings were submitted to moderate and high hyperosmotic stresses, several protein kinases were activated with profiles comparable with those observed in cells (Fig. 1C). Using MBP as a substrate, only the 42-, 38-, and 37-kDa activity bands were visualized, whereas on histone, the four non-MAPK protein kinases were detected. Unlike in cells, kinase activations hardly increased in plantlets submitted to high hyperosmolarity in comparison with moderate stress. This result suggests that signaling induced by high hyperosmotic medium on seedlings (1 m sucrose) corresponds only to signaling induced by a moderate hyperosmotic condition on cell suspension (500 mm sucrose). Coherently, the 44 kDa displayed a strong activation in plantlets exposed to high stress, whereas the activity of this kinase decreased in cell suspension when the stress strength increased. As in cells, the 44-kDa kinase corresponds to At-MPK6 (data not shown). Thus, the four non-MAPK protein kinases are a
Год издания: 2004
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: Plant Stress Responses and Tolerance, Plant-Microbe Interactions and Immunity, Plant nutrient uptake and metabolism
Другие ссылки: Journal of Biological Chemistry (PDF)
Journal of Biological Chemistry (HTML)
HAL (Le Centre pour la Communication Scientifique Directe) (HTML)
hal.science (HTML)
Journal of Biological Chemistry (HTML)
HAL (Le Centre pour la Communication Scientifique Directe) (HTML)
hal.science (HTML)
Открытый доступ: hybrid
Том: 279
Выпуск: 40
Страницы: 41758–41766