CRK2 Enhances Salt Tolerance by Regulating Callose Deposition in Connection with PLDα1

Abstract

High salinity is an increasingly prevalent source of stress to which plants must adapt. The receptor-like protein kinases, including members of the Cys-rich receptor-like kinase (CRK) subfamily, are a highly expanded family of transmembrane proteins in plants that are largely responsible for communication between cells and the extracellular environment. Various CRKs have been implicated in biotic and abiotic stress responses; however, their functions on a cellular level remain largely uncharacterized. Here we have shown that CRK2 enhances salt tolerance at the germination stage in Arabidopsis (Arabidopsis thaliana) and also modulates root length. We established that functional CRK2 is required for salt-induced callose deposition. In doing so, we revealed a role for callose deposition in response to increased salinity and demonstrated its importance for salt tolerance during germination. Using fluorescently tagged proteins, we observed specific changes in the subcellular localization of CRK2 in response to various stress treatments. Many of CRK2's cellular functions were dependent on phospholipase D activity, as were the subcellular localization changes. Thus, we propose that CRK2 acts downstream of phospholipase D during salt stress, promoting callose deposition and regulating plasmodesmal permeability, and that CRK2 adopts specific stress-dependent subcellular localization patterns that allow it to carry out its functions.

CRK2 and C-terminal Phosphorylation of NADPH Oxidase RBOHD Regulate Reactive Oxygen Species Production in Arabidopsis

“…Expression of yellow fluorescent protein (YFP)-tagged CRK2 under the control of the CRK2 promoter (CRK2pro:CRK2-YFP) in crk2 background-restored plant growth (Figures 1B,1C,and Supplemental Figure 1A). Substitution of the ATP binding Lys (K) at position 353 with Glu (E; CRK2 K353E ) or the Asp (D) at position 450 in the catalytic domain VIb (Stone and Walker, 1995) with Asn (N; CRK2 D450N ) abated the kinase activity of CRK2 in vitro (Hunter et al, 2019). Kinase-dead CRK2 K353E -YFP or CRK2 D450N -YFP under control of the CRK2 promoter was expressed in leaves and roots and displayed the same subcellular localization as wild-type CRK2-YFP (Supplemental Figures 1C and 1D) but failed to restore the growth defect of crk2 ( Figures 1B, 1C, and Supplemental Figure 1A).…”

“…CRKs have been linked to ROS signaling (Idänheimo et al, 2014;Bourdais et al, 2015;Yeh et al, 2015;Yadeta et al, 2017) and cell death (Bourdais et al, 2015;Yadeta et al, 2017). CRKs are important signaling elements in plant development and biotic and abiotic stress responses (Chen et al, 2004;Acharya et al, 2007;Wrzaczek et al, 2010;Tanaka et al, 2012;Idänheimo et al, 2014;Bourdais et al, 2015;Yeh et al, 2015;Chern et al, 2016;Yadeta et al, 2017;Hunter et al, 2019).…”

CRK2 and C-terminal phosphorylation of NADPH oxidase RBOHD regulate ROS production in Arabidopsis

“…However, unlike in 279 the response to flg22, salt-induced callose deposition is reduced in crk2 (Hunter et al, 2019) suggesting that 280 CRK2 might regulate different callose synthases in response to biotic and abiotic stimuli. Interestingly, 281 CRK2 forms clusters at the plasma membrane in response to flg22-treatment and ROS is required for this 282 process (Hunter et al, 2019). It is not clear how these clusters are integrated with the regulation of RBOHD 283 activity but it might serve to connect RBOHD-dependent ROS production with callose deposition.…”

“…Thus, the diminished increase of cytosolic Ca 2+ in crk2 may be a consequence of the impaired 276 flg22-induced ROS production. Also, callose deposition (Caillaud et al, 2014;Ellinger & Voigt, 2014) has 277 been previously linked to ROS production (Couto & Zipfel, 2016) and CRK2 interacts with callose synthases 278 and phosphorylates CALLOSE SYNTHASE 1 (CALS1) in vitro (Hunter et al, 2019). However, unlike in 279 the response to flg22, salt-induced callose deposition is reduced in crk2 (Hunter et al, 2019) suggesting that 280 CRK2 might regulate different callose synthases in response to biotic and abiotic stimuli.…”

“…Also, callose deposition (Caillaud et al, 2014;Ellinger & Voigt, 2014) has 277 been previously linked to ROS production (Couto & Zipfel, 2016) and CRK2 interacts with callose synthases 278 and phosphorylates CALLOSE SYNTHASE 1 (CALS1) in vitro (Hunter et al, 2019). However, unlike in 279 the response to flg22, salt-induced callose deposition is reduced in crk2 (Hunter et al, 2019) suggesting that 280 CRK2 might regulate different callose synthases in response to biotic and abiotic stimuli. Interestingly, 281 CRK2 forms clusters at the plasma membrane in response to flg22-treatment and ROS is required for this 282 process (Hunter et al, 2019).…”