SNF1-Related Protein Kinases SnRK2.4 and SnRK2.10 Modulate ROS Homeostasis in Plant Response to Salt Stress

Abstract

In response to salinity and various other environmental stresses, plants accumulate reactive oxygen species (ROS). The ROS produced at very early stages of the stress response act as signaling molecules activating defense mechanisms, whereas those produced at later stages in an uncontrolled way are detrimental to plant cells by damaging lipids, DNA, and proteins. Multiple systems are involved in ROS generation and also in ROS scavenging. Their level and activity are tightly controlled to ensure ROS homeostasis and protect the plant against the negative effects of the environment. The signaling pathways responsible for maintaining ROS homeostasis in abiotic stress conditions remain largely unknown. Here, we show that in Arabidopsis thaliana, two abscisic acid- (ABA)-non-activated SNF1-releted protein kinases 2 (SnRK2) kinases, SnRK2.4 and SnRK2.10, are involved in the regulation of ROS homeostasis in response to salinity. They regulate the expression of several genes responsible for ROS generation at early stages of the stress response as well as those responsible for their removal. Moreover, the SnRK2.4 regulate catalase levels and its activity and the level of ascorbate in seedlings exposed to salt stress.

Keywords: Arabidopsis thaliana; SnRK2; antioxidant enzymes; ascorbate cycle; hydrogen peroxide; reactive oxygen species; salinity.

Figure 1

SnRK2.4 and SnRK2.10 affect ROS level in plants subjected to salt stress. (A). Leaves of wt plants and snrk2.4 and snrk2.10 mutant lines were subjected to 150 mM NaCl for the indicated time and H₂O₂ was determined using a luminol-based assay. Letters represent statistical differences in respect to the wt plants where a means no significant difference, and b means a significant difference [one way analysis of variance (ANOVA). Error bars represent standard deviation (SD). Three independent biological replicates, each with four samples per data point were performed. Results of all combined experiments are shown. B and C. Roots of five-day-old Arabidopsis seedlings (wt plants and snrk2.4 and snrk2.10 mutant lines) were stained with propidium iodide (PI; 20 µg/mL) and 2′,7′-Dichlorofluorescin diacetate (H₂DCFDA; 30 µg/mL) and then treated for 15 min with 250 mM NaCl in ½ MS (+) or ½ MS only (−). (B) The production of ROS was monitored by imaging of H₂DCFDA fluorescence in the roots using confocal microscopy; BF – bright field image, scale bars =50 µm (C) Fluorescence intensity of H₂DCFDA was calculated from well-defined region of interest (4000 µm²) in the root meristematic zone on each single confocal section; stars represent statistically significant differences in respect to the wt plants (Mann-Whitney U test) where *** p < 0.0001; results represent data collected from at least 30 seedlings/line/conditions where each dot represents the sample value and a dash represents the median of measurements.

Figure 1

SnRK2.4 and SnRK2.10 affect ROS level in plants subjected to salt stress. (A). Leaves of wt plants and snrk2.4 and snrk2.10 mutant lines were subjected to 150 mM NaCl for the indicated time and H₂O₂ was determined using a luminol-based assay. Letters represent statistical differences in respect to the wt plants where a means no significant difference, and b means a significant difference [one way analysis of variance (ANOVA). Error bars represent standard deviation (SD). Three independent biological replicates, each with four samples per data point were performed. Results of all combined experiments are shown. B and C. Roots of five-day-old Arabidopsis seedlings (wt plants and snrk2.4 and snrk2.10 mutant lines) were stained with propidium iodide (PI; 20 µg/mL) and 2′,7′-Dichlorofluorescin diacetate (H₂DCFDA; 30 µg/mL) and then treated for 15 min with 250 mM NaCl in ½ MS (+) or ½ MS only (−). (B) The production of ROS was monitored by imaging of H₂DCFDA fluorescence in the roots using confocal microscopy; BF – bright field image, scale bars =50 µm (C) Fluorescence intensity of H₂DCFDA was calculated from well-defined region of interest (4000 µm²) in the root meristematic zone on each single confocal section; stars represent statistically significant differences in respect to the wt plants (Mann-Whitney U test) where *** p < 0.0001; results represent data collected from at least 30 seedlings/line/conditions where each dot represents the sample value and a dash represents the median of measurements.

Figure 2

SnRK2.4 and SnRK2.10 affect the expression of genes involved in ROS homeostasis during response to salt stress. Expression (mRNA level) of (A). RbohD—respiratory burst oxidase homolog protein D; (B) RbohF—respiratory burst oxidase homolog protein F; (C) PRX33—peroxidase 33; and (D) PRX34—peroxidase 34 was determined by RT-qPCR in wt plants and snrk2 mutant lines subjected to treatment with 150 mM NaCl at times indicated (h); error bars represent SD; stars represent statistically significant differences in comparison with the wt plants (Student t-test) where * p < 0.05; ** p < 0.001; *** p < 0.0001. At least two independent biological replicates of the experiment were performed. Results of one representative experiment are shown.

Figure 3

SnRK2.4 and SnRK2.10 modulate catalase (CAT) on multiple levels during response to salt stress. Wild type and snrk2 mutants’ seedlings were subjected to treatment with 150 mM NaCl for times indicated. CAT1 expression was determined by RT-qPCR (A), total catalase protein was determined by immunoblot analysis (B), and total catalase activity assay was performed (C); error bars represent SD; stars represent statistically significant differences in comparison with the wt plants (Student t-test) where * p < 0.05; ** p < 0.001; *** p < 0.0001. After exposure, membranes were stripped and reused for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) detection as a loading control. At least two independent biological replicates of the experiment were performed, each with four samples per data point. Results of one representative experiment are shown.

Figure 4

SnRK2.4 and SnRK2.10 regulate enzymes of the ascorbate cycle during response to salt stress. Wild type and snrk2 mutant seedlings were subjected to treatment with 150 mM NaCl for times indicated. Expression of (A) APX1—Ascorbate Peroxidase 1, (B) APX2—Ascorbate Peroxidase 2, (C) APX6—Ascorbate Peroxidase 6, and (D) DHAR1—Dehydroascorbate Reductase 1 was monitored by RT-qPCR; error bars represent SD; stars represent statistically significant differences in comparison with the wt plants (Student t-test) where * p < 0.05; ** p < 0.001; *** p < 0.0001. Total protein level of (E) APX and (F) DHAR1 was monitored with immunoblot analysis; after exposure, membranes were stripped and reused for GAPDH detection as a loading control. At least two independent biological replicates of the experiment were performed. Results of one representative experiment are shown.

Figure 5

SnRK2.4 and SnRK2.10 regulate ascorbate cycle during response to salt stress. Wild type and snrk2 mutant seedlings were subjected to treatment with 150 mM NaCl for times indicated and (A) ascorbate content, (B) ascorbate peroxidase (APX) activity, and (C) ascorbate redox status were monitored. Asc—ascorbate, DHAsc—dehydroascorbate; error bars represent SD; stars represent statistically significant differences from wt plants (Student t-test for Asc and APX activity, Chi-square test for Asc/DHAsc ratio) where * p < 0.05; ** p < 0.001; *** p < 0.0001. At least two independent biological replicates of the experiment were performed, each with four samples per data point. Results of one representative experiment are shown.

Figure 6

Possible roles of SnRK2.4 and SnRK2.10 in the regulation of the ROS homeostasis in Arabidopsis seedlings exposed to the salt stress. Proposed role of the SnRK2s in (A) early response and (B) late response to the salt stress. In response to salinity, SnRK2.4 along with SnRK2.10 regulate the ROS production/accumulation as well as ROS scavenging at the transcription as well as protein and/or activity levels. Detailed description in the text; dash lines—SnRK2.4 impact; dotted lines—SnRK2.10 impact; green question mark—probably indirect regulation; red question mark—plausible direct regulation by phosphorylation.

Figure 7

Schematic model illustrating the role of SnRK2.4 and SnRK2.10 in Arabidopsis’ response to salt stress. SnRK2.4 and SnRK2.10 modulate root growth under the salinity conditions. Moreover, in response to salt stress, the ABA-non-activated SnRK2s phosphorylate VARICOSE (VCS), a protein participating in mRNA decay, and two dehydrins, Early Responsive to Dehydration 10 (ERD10) and ERD14. Our results presented here revealed that SnRK2.4 and SnRK2.10 regulate the ROS homeostasis in the response to salinity.