CRK2 Enhances Salt Tolerance by Regulating Callose Deposition in Connection with PLD α 1

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.

Figure 1.

CRK2 enhances salt tolerance. A, Overexpression of CRK2 increases salt tolerance at the germination stage; loss of functional CRK2 reduces salt tolerance. Data were normalized to the untreated controls for each line. Comparisons are to Col-0 (one-way ANOVA, post hoc Dunnett); n = 3; error bars indicate the sd. B, CRK2 is an active kinase in vitro; kinase-dead protein variants lack kinase activity. C and D, CRK2 is involved in primary root elongation under standard growth conditions (C) and in 150 mm NaCl (D). Comparisons are to Col-0 (one-way ANOVA, post hoc Dunnett); 8-d-old seedlings, transplanted to treatments at 5 d; n = at least 16; box limits represent the 25th and 75th percentiles; the horizontal line represents the median; whiskers extend to the minimal and maximal values. ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2.

CRK2 subcellular protein localization. A, CRK2-YFP localizes uniformly to the plasma membrane under standard growth conditions. Arrows indicate the presence of Hechtian strands following plasmolysis. B, In response to abiotic and biotic stresses, 35S::CRK2-YFP relocalizes to distinct stress-specific patterns along the plasma membrane. Images are of epidermal cells from 7-d-old seedlings; stresses applied were as follows: mannitol, 800 mm for 15 min; NaCl, 150 mm for 30 min; flg22, 10 µm for 30 min; H₂O₂, 1 mm for 30 min. C, Colocalization with callose deposits supports NaCl-induced plasmodesmal localization of CRK2-YFP; the white boxes in the upper images indicate the zoomed areas in the lower images. D, CRK2-YFP does not colocalize (arrows) with plasmodesmal marker PDLP5-RFP under standard growth conditions. E, CRK2-YFP partially colocalizes (arrows) with PDLP5-RFP following NaCl treatment. F, Quantification of CRK2-YFP colocalization with callose deposits and PDLP5-RFP following NaCl treatment. Scale bars = 10 µm. G, Quantification of NaCl-induced relocalization of CRK2-YFP by percent enrichment at relocalization domains; box limits represent the 25th and 75th percentiles; the horizontal line represents the median; whiskers extend to the minimal and maximal values; ***P < 0.001 (one-way ANOVA, pooled t test).

Figure 3.

Mechanism of CRK2 stress-dependent localization changes. A, Kinase activity is required for both abiotic and biotic stress-induced relocalization. B, NADPH-dependent ROS production is required for the biotic response, but not for the abiotic relocalization. C, Increased cytosolic calcium is required for both abiotic and biotic relocalization. D, Increased cytosolic calcium is sufficient to induce CRK2 relocalization: i, dimethyl sulfoxide (DMSO) control; ii, CaCl₂; iii, CaCl₂ + ionomycin; iv, CPA; v, DPI + CaCl₂ + ionomycin; vi, DPI + CPA; vii, CRK2^K353E + CaCl₂ + ionomycin. E, Clathrin-mediated internalization is not required for either abiotic or biotic relocalization. F and G, PLD activity is required for both abiotic and biotic relocalization. H, PLDɑ1 is required for both abiotic and biotic relocalization. 35S overexpression lines were used in all replicates; images are epidermal cells from 7-d-old seedlings; treatment times and conditions are according to Table 2. Scale bars = 10 µm.

Figure 4.

CRK2 is required for salt-induced callose deposition. A, Aniline blue staining for callose deposition. Scale bars = 100 µm. B, Kinase-active CRK2 is required for NaCl-induced callose deposition. The graph shows quantification of callose deposits; comparisons are between untreated and NaCl-treated samples for each line (one-way ANOVA, post hoc Tukey’s HSD mean-separation test); n = at least 15. C, Germination of the cals1 mutant is reduced on salt-containing media. Comparisons are to Col-0 (one-way ANOVA, post hoc Dunnett); error bars indicate the sd; n = 3. D, CALS1 is required for NaCl-induced callose deposition. Comparisons are between untreated and NaCl-treated samples (one-way ANOVA, post hoc Tukey’s HSD mean-separation test); n = at least 6. E, Impact of PLD on callose deposition in CRK2 lines. Comparisons are between untreated and NaCl-treated samples pretreated with 1-butanol or 2-butanol for each line (one-way ANOVA, post hoc Tukey’s HSD mean-separation test); n ≥ 6. F, CRK2 can phosphorylate the N terminus of CALS1 in vitro but cannot phosphorylate PLDα1. G, Plasmodesmal permeability during standard growth conditions. The observed callose deposition correlates with changes in plasmodesmal permeability. Quantification by percent diffusion of a fluorescent intracellular dye from the adaxial to the abaxial surface; comparisons are to Col-0 (one-way ANOVA, post hoc Dunnett); n = 3. Seedlings were 7 d old; the treatment protocol was NaCl, 150 mm for 30 min; box limits represent the 25th and 75th percentiles; the horizontal line represents the median; whiskers extend to the minimal and maxal values; ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5.

CRK2 is not required for the initial salt-induced calcium response. A, Fluo-4-AM calcium imaging of cell-level Ca²⁺ influx in response to 150 mm NaCl in epidermal cells of 7-d-old seedlings; n = 3; ∼70 cells were measured per replicate. Scale bars = 100 µm. B, YCNano-65 calcium imaging of tissue-level Ca²⁺ influx in response to 150 mm NaCl; 7-d-old seedlings; n ≥ 6. Scale bars = 1 mm. Additions were made at t = 0. Error bars indicate the se.

Figure 6.

Schematic of the proposed pathway for CRK2 regulation of callose deposition at plasmodesmata during salt stress. A, Resting state. B, Early responses to salt stress. Increased extracellular NaCl triggers Ca²⁺ influx. Cytoplasmic Ca²⁺ elevation activates PLDα1 leading to PA production and a shift in membrane properties; this serves as a scaffold for changes in CRK2 localization from uniformly along the plasma membrane to specific domains concentrated at plasmodesmata. Once localized at plasmodesmata, CRK2 interacts with CALS1 to promote callose deposition, ultimately leading to enhanced salt tolerance.