Identification of Nine Sucrose Nonfermenting 1-related Protein Kinases 2 Activated by Hyperosmotic and Saline Stresses in Arabidopsis thaliana

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. 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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. 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Plant Cell. 2001; 13: PubMed Scopus Google Scholar). have reported the activation of several protein kinases that not for 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 In we demonstrated that these kinases were also activated in Arabidopsis seedlings and to the SnRK2 identification that nine of the 10 SnRK2 were activated by Then the possible involvement of SnRK2 in transduction pathways such as saline cold, and abscisic acid was also Plant cell suspension was in as described (1Droillard M.J. Boudsocq M. Barbier-Brygoo H. Laurière C. FEBS Lett. 2002; 527: 43-50Crossref PubMed Scopus (146) Google Scholar) in were used with of cell A. were and on a 2 MES, and T. F. Physiol. Scopus Google Scholar), 10 and The seedlings were with a and of was using the in I. The of and were as a and the of and as a of the were by were expressed in E. and using to the used for SnRK2 and of and reverse in a of of SnRK2 were by using and that were The of the are in the reverse the was to In vitro using as were to the The of was used to E. and the were by used for SnRK2 of and reverse in a to to were to the using as The of was used to E. of was in the of the of A. R. J. J. C. Plant J. 2000; PubMed Google Scholar) in the with I. The was in the of the expression of the of a a was cell suspension and seedlings using Plant The was of and using the was for using the in I. were on and by The of the was by and of the and of an the of and was with the and expression was shown to be J.M. S. S. Plant J. 1996; 10: PubMed Scopus Google Scholar). was using a were for in 10 and to with was by the of or hyperosmotic or or In the sucrose was by mannitol or NaCl to treatment, cell suspension was in and were for in the described were to or to the or sucrose for treatment, seedlings were in and of were used 3 for of were by for and in of in which not sucrose but and was to with and cells were for 3 on a in were by for with and in for were in were with of and of mannitol, an in the for the was with and for were in of and in the for osmotic were for and in the of or hyperosmotic mannitol with mannitol, or NaCl with NaCl, for 10 cold treatment, were for 10 or for the treatment, were for with or the of were for and the was in of or seedlings were in and in 2 10 DTT, for the was in acid 10 with cold and in was by the PubMed Scopus Google Scholar). the was as in in the of and protein to with and NaCl. The was on with of and was in and was on with myelin basic protein or as for the The were for protein as described by Zhang et al. S. H. Klessig D.F. Plant Cell. 1998; 10: Google Scholar). the the were for in kinase 2 DTT, was for in of the with cold and of Then the were in acid and The protein kinase was on the by the were with the antibody or with a plant kinase was raised against of the of SnRK2 kinases cells or was with of or of in 10 DTT, NaCl, for 3 Then of protein was and was for The was in and in kinase 2 2 DTT, and in of was by the kinase as was with different the SnRK2 used for and one The antibody was for 10 with the protein and The was used a of or or were on and were with in NaCl, and with or or were used as and the were using the and enhanced for the described in are as and Several by and in Arabidopsis and the activation of protein kinases in response to hyperosmotic kinase were in the of using or as or hyperosmotic stresses were in to an as described (1Droillard M.J. Boudsocq M. Barbier-Brygoo H. Laurière C. FEBS Lett. 2002; 527: 43-50Crossref PubMed Scopus (146) Google Scholar). Arabidopsis cell suspension was to hyperosmolarity, the activation of several kinases was observed using as a with molecular of and and a one The were the stress was the were using as a a activation was with with molecular of and in to the and protein kinases activated by hyperosmolarity both and MBP, indicating that not belong to the MAPK contrast, the kinase on but not on has 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 of mannitol and NaCl, was in leading to the cells were with mannitol, or NaCl, activation profiles were indicating that these kinase a response to osmotic stress. Arabidopsis seedlings were to and hyperosmotic several protein kinases were activated with profiles with observed in cells Using as a only the and were whereas on the non-MAPK protein kinases were in kinase in to hyperosmolarity in with stress. that signaling induced by hyperosmotic on seedlings only to signaling induced by a hyperosmotic on cell suspension the displayed a activation in exposed to whereas the of kinase in cell suspension the stress in the kinase to not the non-MAPK protein kinases are activated both in cells and seedlings, that Arabidopsis cell suspension is a suitable to these kinases in response to osmotic The by in Arabidopsis non-MAPK protein kinases activated by hyperosmotic stresses in cells and seedlings remained to be identified. was assumed that belong to the Arabidopsis SnRK2 family and the expression of the 10 of the family was Using the of SnRK2 was in cells and seedlings the used for kinase assays. 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FEBS Lett. 2002; 527: 43-50Crossref PubMed Scopus (146) Google Scholar) is not by be that the SnRK2 antibody whereas only of them were by on be by different expression activation one SnRK2 to the using the family-specific antibody that SnRK2 kinases are activated by hyperosmolarity in Arabidopsis cell stress activates several SnRK2 kinases in Arabidopsis cell suspension. were as described in the to of the or of the SnRK2 was with an kinase the of the the SnRK2 used for or an kinase were as of Nine SnRK2 by identification the molecular of the activated SnRK2 was investigated using a transient expression Arabidopsis be cell have transduction we by kinase that SnRK2 kinases were also activated by hyperosmolarity in Arabidopsis were to or mannitol hyperosmotic as for In such several kinases were activated in with the as in cells Using the antibody in by kinase a activation of the SnRK2 and was observed in hyperosmotic being a suitable to the activated SnRK2, Arabidopsis were with an expression for each SnRK2 or the as a were for mannitol hyperosmotic stress as described with SnRK2 was by kinase using as a The expression of each SnRK2 was by with antibody SnRK2 were activated by hyperosmotic stress. activation by hyperosmolarity be a activation of a activation of and and a activation of and SnRK2 by and but by pathways of stresses with between salt, cold, and signaling Schumaker Zhu J.K. Plant Cell. 2002; PubMed Scopus Google Scholar). nine of 10 SnRK2 kinases are activated by hyperosmotic the possible involvement of these kinases in transduction pathways was for hyperosmotic the activation of SnRK2 kinases by salt cold, or was in with the family-specific salt were to or NaCl hyperosmotic cold treatment, were or as a treatment, were with or the of Using the antibody in by kinase SnRK2 of and were activated by salt as observed with mannitol hyperosmotic whereas only activated SnRK2 of and and a kinase. the cold not SnRK2 kinase the stress was to not different SnRK2 were activated by salt stress and ABA, the transient expression was to the SnRK2 activated by each stress and were as described SnRK2 and expression of each kinase were as stress activated the kinases as mannitol and with the for which is activated by NaCl mannitol is with the kinase activation observed in response to mannitol, and NaCl in Arabidopsis cell suspension was that and a on the with hyperosmotic and saline only activated five SnRK2 for mannitol treatment, a activation of a activation of and and a activation of were was only activated by ABA, to the activation by hyperosmotic stress signaling different protein kinase families and them the MAPK family is the in plants. we the activation by hyperosmotic stresses of several Arabidopsis protein kinases with molecular of and kinases both and as and was shown that activation was to a MAPK and to a (1Droillard M.J. Boudsocq M. Barbier-Brygoo H. Laurière C. FEBS Lett. 2002; 527: 43-50Crossref PubMed Scopus (146) Google Scholar). these that hyperosmotic stress activates protein kinases that not belong to the MAPK family in Arabidopsis cell suspension. Moreover, were induced by different such as mannitol, and NaCl and 2 of stress (1Droillard M.J. Boudsocq M. Barbier-Brygoo H. Laurière C. FEBS Lett. 2002; 527: 43-50Crossref PubMed Scopus (146) Google Scholar), indicating that these kinases are involved in a and response to osmotic stress. Similar kinase activation profiles were observed cell seedlings and or were exposed to hyperosmolarity, indicating that cells and response in plants, for the transduction be that of the kinases was different on plant which be of in the of protein The kinase activation was also in in to cell suspension. Moreover, the of kinase activation in cell to stress and seedlings exposed to stress between cells and plant was also reported for the phosphorylation of the protein induced by the hyperosmotic in tobacco K. Plant Cell. 2001; 13: PubMed Scopus Google Scholar). The activation for a stress in cells in to and the by the to a osmotic stress to cells using cell suspension and to be suitable to plant osmotic on the activation of one SnRK2 by hyperosmolarity in tobacco cells M. Awotunde O.S. Muszynska G. Klessig D.F. Dobrowolska G. Plant Cell. 2000; 12: 165-178Crossref PubMed Scopus (261) Google Scholar), the non-MAPK protein kinases in Arabidopsis were suggested to belong to the SnRK2 is known the of family, on the expression or M. N. J. Luan S. M. K. Zhu J.K. A.C. Plant Physiol. PubMed Scopus Google for The 10 Arabidopsis SnRK2 were expressed both in cells and seedlings 2), and the whole kinase family a suitable candidate. the involvement of SnRK2 in osmotic a family-specific antibody was with that the antibody the 10 SnRK2 but none of the 10 Moreover, the antibody only on the protein cell suspension with molecular and to SnRK2 in indicating that the antibody was to the SnRK2 Using antibody in with kinase the activation of SnRK2 was The SnRK2 displayed the molecular as observed in suggesting that are the is the of activation by hyperosmotic stresses of several SnRK2 in indicating an important role of family in osmotic signaling. the SnRK2 kinases the molecular a transient expression was Using a the of the kinases with transduction pathways and not the expression of the kinases are by the sensitive not In the transient expression mannitol hyperosmotic stress activated nine of the 10 SnRK2 kinases be that nine SnRK2 were whereas only were observed in SnRK2 kinases have molecular which is with on as in on molecular a between in and SnRK2 be The to whereas the and The activated SnRK2 and the SnRK2 were activated with different activation suggesting that play a role to activation are with that activation of SnRK2. The tobacco activated by hyperosmotic stress M. Awotunde O.S. Muszynska G. Klessig D.F. Dobrowolska G. Plant Cell. 2000; 12: 165-178Crossref PubMed Scopus (261) Google Scholar) and the Arabidopsis to R. T. Ichimura K. Mizoguchi T. F. J. Shinozaki K. Plant Physiol. 2002; PubMed Scopus Google Scholar) to and these kinases are shown to be activated by hyperosmotic and saline In et al. K. Plant Cell. 2001; 13: PubMed Scopus Google Scholar) described the activation of only of SnRK2 and in yeast exposed to In the et al. Y. S. H. Y. T. Plant Cell. PubMed Scopus Google Scholar) described the salt activation of the 10 SnRK2 by transient expression assays. of them and were also shown to be activated by mannitol but not by In the the activation of the whole family by different osmotic was in with the and cold not of the 10 Arabidopsis SnRK2 whereas salt stress activated the nine SnRK2 mannitol stress that SnRK2 are activated in a osmotic response as observed in cells which was the only SnRK2 not to be activated by of the NaCl, cold, and is the protein of the family displayed expression in the SnRK2 and and the be by a protein the SnRK2 activation expressed not be involved in transduction pathway and on protein expression in plant Interestingly, only activated a of the nine SnRK2 but with activation in the the kinases are activated by stresses cold, and the of the be that activation observed in transient expression the in on SnRK2 be in one composed of kinases activated by both and hyperosmotic including and and the one composed of kinases only activated by hyperosmotic including and is with the in of the which was transcriptionally induced by A. 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Shinozaki K. Plant Cell. 2001; 13: PubMed Scopus Google Scholar). nine SnRK2 are activated by hyperosmotic be to in the pathway. was shown that SnRK2 displayed different activation and only five of the nine kinases were also activated by ABA, suggesting that be involved in several signaling of expression in the and of the be to for upstream and the of the kinases a is on possible the of in that are to be are to the of SnRK2 and in the cell stresses to the one kinase activation is phosphorylation by upstream SnRK2, were using by or of The tobacco M. Awotunde O.S. Muszynska G. Klessig D.F. Dobrowolska G. Plant Cell. 2000; 12: 165-178Crossref PubMed Scopus (261) Google Scholar) and SnRK2 Y. S. H. Y. T. Plant Cell. PubMed Scopus Google Scholar) were shown to be activated by In contrast, and Zhang M.E. Zhang S.Q. Plant Physiol. 2000; 122: 1355-1363Crossref PubMed Scopus (107) Google Scholar) reported activation by hyperosmolarity of an kinase which is to also be an SnRK2, a is in the for the whole Arabidopsis SnRK2 for the and for the of