Phosphorylation by AtMPK6 is required for the biological function of AtMYB41 in Arabidopsis My Hanh Thi Hoang a , Xuan Canh Nguyen a , Kyunghee Lee b,1 , Young Sang Kwon a , Huyen Trang Thi Pham a , Hyeong Cheol Park a,b , Dae-Jin Yun a,b , Chae Oh Lim a,b , Woo Sik Chung a,b,⇑ a Division of Applied Life Science (BK21 Program), Gyeongsang National University, Jinju 660-701, Republic of Korea b Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Republic of Korea article info Article history: Received 18 April 2012 Available online 30 April 2012 Keywords: Arabidopsis MAP kinase Phosphorylation Transcription factor Salt abstract Mitogen-activated protein kinases (MPKs) are involved in a number of signaling pathways that control plant development and stress tolerance via the phosphorylation of target molecules. However, so far only a limited number of target molecules have been identified. Here, we provide evidence that MYB41 rep- resents a new target of MPK6. MYB41 interacts with MPK6 not only in vitro but also in planta. MYB41 was phosphorylated by recombinant MPK6 as well as by plant MPK6. Ser 251 in MYB41 was identified as the site phosphorylated by MPK6. The phosphorylation of MYB41 by MPK6 enhanced its DNA binding to the promoter of a LTP gene. Interestingly, transgenic plants over-expressing MYB41 WT showed enhanced salt tolerance, whereas transgenic plants over-expressing MYB41 S251A showed decreased salt tolerance dur- ing seed germination and initial root growth. These results indicate that the phosphorylation of MYB41 by MPK6 is required for the biological function of MYB41 in salt tolerance. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction MPK cascades are signal transduction pathways that are highly conserved and widespread in all eukaryotic cells, including yeasts, animals and plants [1–3]. MPKs play a central role for converting extracellular signals, including environmental stresses, into internal signal transduction and activation of intracellular responses [3–6]. It is also well documented that plant MPKs are activated by a variety of environmental stimuli including salt, cold, wounding, heat, os- motic shock, heavy metal, UV, drought and pathogen attack [1,2,7,8]. In Arabidopsis, MPK4 and MPK6 are activated by high salt [9]. MPK6-null mutant seedlings were more salt sensitive than wild type [10]. Over-expression of MKK2 generated salt tolerance through the constitutive activation of MPK4 and MPK6 [11]. Fur- thermore, MKK2 interacted with an upstream MKKK, MEKK1, is activated by high salt [12,13]. These studies demonstrated that the MPK cascade consisting of MEKK1–MKK2–MPK6 plays a role in salt tolerance. However, the direct downstream targets of MPK6 that is involved in salt tolerance signaling pathway are unknown. The MYB transcription factors represent one of the most com- plex transcription factor families composed of approximately 125 members in Arabidopsis [14]. MYBs are known to be involved in the regulation of plant development and stress responses [15,16]. Among them, AtMYB41 has originally been identified as a tran- scription factor controlling cell expansion and cuticle deposition in response to abiotic stress [17,18]. However, a post-translational regulation of MYB41 has not been elucidated in response to salt stress. Here, we report that AtMYB41 is phosphorylated and acti- vated by MPK6 in response to salt stress. Interestingly, we showed that the phosphorylation of MYB41 by MPK6 is required for in- creased salt tolerance in Arabidopsis. 2. Materials and methods 2.1. Plant materials and growth conditions Wild type (Arabidopsis thaliana, ecotype Columbia), MYB41 WT and MYB41 S251A over-expressing transgenic, mpk3-2 (salk_ 151594), mpk6-3 (salk_127507) mutant plants were grown in a controlled culture chamber at 22 °C with a 16 h light/8 h dark cycle at a light intensity of 100 Em 2 S 1 . To activate MPK6, WT, mpk3-2 and mpk6-3 plants were treated with 150 mM NaCl for 30 min. 2.2. Pull-down assay Approximately 5 lg of GST–MYB41 was bound to glutathione beads in binding buffer (20 mM Tris–HCl, pH 7.5; 200 mM NaCl; 1% Triton X-100; 0.1 mM EDTA; 0.5 mM DTT) for 2 h at 4 °C. The 0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2012.04.137 ⇑ Corresponding author at: Division of Applied Life Science (BK21 Program), Gyeongsang National University, Jinju 660-701, Republic of Korea. Fax: +82 55 759 9363. E-mail address: chungws@gnu.ac.kr (W.S. Chung). 1 Present address: College of Pharmacy, Yeungnam University, Daegu, Republic of Korea. Biochemical and Biophysical Research Communications 422 (2012) 181–186 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc