1 3 The Arabidopsis thaliana cysteine-rich receptor-like kinases CRK6 and 4 CRK7 protect against apoplastic oxidative stress 5 6 7 Niina Idänheimo a,1 Q1 , Adrien Gauthier a,1 , Jarkko Salojärvi a , Riccardo Siligato a,b , Mikael Brosché a,c , 8 Hannes Kollist c , Ari Pekka Mähönen a,b , Jaakko Kangasjärvi a , Michael Wrzaczek a,⇑ 9 a Division of Plant Biology, Department of Biosciences, University of Helsinki, FI-00014 Helsinki, Finland 10 b Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland 11 c Institute of Technology, University of Tartu, Tartu 50411, Estonia 12 13 15 article info 16 Article history: 17 Received 3 February 2014 18 Available online xxxx 19 Keywords: 20 CRK 21 RLK 22 ROS signaling 23 Oxidative stress 24 Redundancy 25 Arabidopsis thaliana 26 27 abstract 28 Receptor-like kinases are important regulators of many different processes in plants. Despite their large 29 number only a few have been functionally characterized. One of the largest subgroups of receptor-like 30 kinases in Arabidopsis is the cysteine-rich receptor like kinases (CRKs). High sequence similarity among 31 the CRKs has been suggested as major cause for functional redundancy. The genomic localization of 32 CRK genes in back-to-back repeats has made their characterization through mutant analysis unpractical. 33 Expression profiling has linked the CRKs with reactive oxygen species, important signaling molecules in 34 plants. Here we have investigated the role of two CRKs, CRK6 and CRK7, and analyzed their role in extra- 35 cellular ROS signaling. CRK6 and CRK7 are active protein kinases with differential preference for divalent 36 cations. Our results suggest that CRK7 is involved in mediating the responses to extracellular but not 37 chloroplastic ROS production. 38 Ó 2014 Published by Elsevier Inc. 39 40 41 42 1. Introduction 43 Plants have developed sophisticated signaling systems for envi- 44 ronment-to-cell and cell-to-cell communication to cope with 45 changing environmental conditions. Biotic and abiotic environ- 46 mental cues can induce specific signaling pathways and trigger 47 downstream signal transmission. In addition to external stimuli, 48 plants use intra- and intercellular messenger molecules including 49 peptides, hormones but also reactive oxygen species (ROS) for 50 the control of growth, development and survival [1,2]. 51 ROS are highly reactive oxygen-based molecules that include 52 hydrogen peroxide (H 2 O 2 ), hydroxyl radical (HO  ), singlet oxygen 53 ( 1 O 2 ) and superoxide ðO  2 Þ. ROS are constantly produced as by- 54 products of cellular metabolism. Due to their ability to oxidize a 55 wide range of biomolecules, ROS levels are tightly controlled to 56 avoid cellular damage [2]. The role of ROS in signal transduction 57 is currently not well understood but recent research has identified 58 redox modification of target proteins and lipids as one major 59 mechanism [2]. However, despite the recent advances in ROS re- 60 search the perception of extracellular ROS is still unclear. 61 Several distinct systems have been proposed to be involved in 62 sensing ROS in the plant extracellular space [2–6]. Changes in 63 extracellular ROS levels affect the ascorbate gradient and lead to 64 changes in cellular redox homeostasis [7,8]. Superoxide, produced 65 by NADPH oxidases, is dismutated to H 2 O 2 which can cross plasma 66 membranes through aquaporins [9]. ROS can cause direct redox 67 modifications to secreted and membrane-localized proteins and 68 lipids. These changes could be sensed through conformational 69 changes or breakdown products, which might act as ligands for 70 receptors. Receptor-like protein kinases (RLKs), one group of po- 71 tential receptors, are membrane proteins that participate in many 72 important signaling processes including plant growth and develop- 73 ment, hormone signaling, and stress responses. A growing body of 74 evidence suggests that a significant number of RLKs is involved in 75 the response to environmental cues [10,11]. The more than 600 76 RLKs encoded in the Arabidopsis genome are divided into subfam- 77 ilies according to their extracellular domain [12]. The variable 78 extracellular domain is typically responsible for signal perception 79 but also protein–protein interaction; the variety ensures percep- 80 tion of a wide range of signals. http://dx.doi.org/10.1016/j.bbrc.2014.02.013 0006-291X/Ó 2014 Published by Elsevier Inc. ⇑ Corresponding author. Address: Division of Plant Biology, Department of Biosciences, University of Helsinki, Viikinkaari 1 (POB65), FI-00014 Helsinki, Finland. Fax: +358 9 191 59552. E-mail addresses: niina.idanheimo@helsinki.fi (N. Idänheimo), adrien.gauthier@ helsinki.fi (A. Gauthier), jarkko.salojarvi@helsinki.fi (J. Salojärvi), riccardo.siligato@ helsinki.fi (R. Siligato), mikael.brosche@helsinki.fi (M. Brosché), hannes.kollist@ ut.ee (H. Kollist), aripekka.mahonen@helsinki.fi (A.P. Mähönen), jaakko.kangasjarvi @helsinki.fi (J. Kangasjärvi), michael.wrzaczek@helsinki.fi (M. Wrzaczek). 1 These authors contributed equally to this work. Biochemical and Biophysical Research Communications xxx (2014) xxx–xxx Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc YBBRC 31637 No. of Pages 6, Model 5G 15 February 2014 Please cite this article in press as: N. Idänheimo et al., The Arabidopsis thaliana cysteine-rich receptor-like kinases CRK6 and CRK7 protect against apoplastic oxidative stress, Biochem. Biophys. Res. Commun. (2014), http://dx.doi.org/10.1016/j.bbrc.2014.02.013