Salinity Stress-Mediated Suppression of Expression of Salt Overly Sensitive Signaling Pathway Genes Suggests Negative Regulation by AtbZIP62 Transcription Factor in Arabidopsis thaliana

Abstract

Salt stress is one of the most serious threats in plants, reducing crop yield and production. The salt overly sensitive (SOS) pathway in plants is a salt-responsive pathway that acts as a janitor of the cell to sweep out Na⁺ ions. Transcription factors (TFs) are key regulators of expression and/or repression of genes. The basic leucine zipper (bZIP) TF is a large family of TFs regulating various cellular processes in plants. In the current study, we investigated the role of the Arabidopsis thaliana bZIP62 TF in the regulation of SOS signaling pathway by measuring the transcript accumulation of its key genes such as SOS1, 2, and 3, in both wild-type (WT) and atbzip62 knock-out (KO) mutants under salinity stress. We further observed the activation of enzymatic and non-enzymatic antioxidant systems in the wild-type, atbzip62, atcat2 (lacking catalase activity), and atnced3 (lacking 9-cis-epoxycarotenoid dioxygenase involved in the ABA pathway) KO mutants. Our findings revealed that atbzip62 plants exhibited an enhanced salt-sensitive phenotypic response similar to atnced3 and atcat2 compared to WT, 10 days after 150 mM NaCl treatment. Interestingly, the transcriptional levels of SOS1, SOS2, and SOS3 increased significantly over time in the atbzip62 upon NaCl application, while they were downregulated in the wild type. We also measured chlorophyll a and b, pheophytin a and b, total pheophytin, and total carotenoids. We observed that the atbzip62 exhibited an increase in chlorophyll and total carotenoid contents, as well as proline contents, while it exhibited a non-significant increase in catalase activity. Our results suggest that AtbZIP62 negatively regulates the transcriptional events of SOS pathway genes, AtbZIP18 and AtbZIP69 while modulating the antioxidant response to salt tolerance in Arabidopsis.

Keywords: Arabidopsis; AtbZIP62; SOS signaling pathway; salt tolerance; transcription regulation.

Figure 1

Growth of Arabidopsis mutant lines on gradient NaCl concentrations. Four Arabidopsis genotypes, the Col-0 wild type, the atbzip62, atnced3, and atcat2 loss-of-function mutants were exposed to salt stress induced by gradient NaCl concentrations. The survival of all genotypes was recorded as a percentage of seedlings having green cotyledons over the total number of seeds germinated (referred to as cotyledon development frequency or CDF) on half-strength MS medium for two weeks. Data points are the mean values of triplicate assays while bars indicate ±SD. The statistical significance level is indicated by asterisks on top of each bar. *** p < 0.05 compared to the wild type; ns, non-significant.

Figure 2

Phenotypic response of Arabidopsis genotypes to salt stress at the rosette stage 10 days after NaCl treatment. The left panel shows plants and detached leaves grown under normal conditions (no salt treatment), whereas the right panel shows plants subjected to salt stress conditions (150 mM). Pictures were taken 10 days after salt stress induction. Adobe Photoshop CS6 was used to remove the background for better display and visualization.

Figure 3

Transcripts accumulation of AtbZIP transcription factors (TFs) encoding genes, AtPYD1, SOS, and glutamate synthase under salt stress. (A) AtPYD1 expression in response to salt stress (150 mM) for 3 to 6 h in Col-0 wild type, atbzip62 loss-of-function mutant, and atcat2. (B) AtbZIP62 transcript accumulation in Col-0, atbzip62 loss-of-function mutant, and atcat2. (C) Expression pattern of AtbZIP18 over time. (D) Expression pattern of AtbZIP69 over time. (E) Expression of SOS1 in Col-0 wild type and atbzip62 loss-of-function mutant over time. (F) Transcriptional level of SOS2, (G) SOS3, (H) AtGLS1, and (I) AtGLS2 overtime at the rosette stage in Col-0 and atbzip62. All the data points are the mean of triplicate assays, while error bars represent ± SD. Statistical significance is indicated by asterisks on top of bars. ** p < 0.01; *** p < 0.001 compared to wild type; ns, non-significant.

Figure 4

Pattern of chloroplast pigment content under salt stress. (A) Chlorophyll a content. (B) Chlorophyll b content. (C) Chlorophyll a + b content. (D) Total carotenoids. (E) Pheophytin a content. (F) Pheophytin b content. (G) Pheophytin a + b content. (H) Total carotenoids relative to pheophytin a and b (Cx + c). The first set of three bars in each panel represents time 0 h (no salt stress), the second set of three bars represents enzyme activity at 3 h post-stress induction, and the third set of three bars represents enzyme activity at 6 h post-stress induction. All the data points are the mean of triplicate assays, while error bar represents ± SD. The asterisks on top of bars are statistical significance level with 95% confidence level. * p < 0.05; *** p < 0.001 compared to wild type; ns, non-significant.

Figure 5

Antioxidant enzyme activity in response to 150 mM NaCl in Arabidopsis genotypes (Col-0 wild type, atbzip62, atnced3, and atcat2 mutants) subjected to salt stress conditions at the rosette stage. (A) Catalase (CAT) activity. (B) Peroxidase (POD) activity. (C) Polyphenol oxidase (PPO) activity. (D) Change in total protein content. (E) Change in proline content. Data in A–E were collected 3 h and 6 h after stress. (F) Malondialdehyde (MDA) content as a measure of lipid peroxidation for Col-0 wild type, atbzip62, and atnced3 exposed to no salt or to 150 mM NaCl. The data points are mean values of triplicate assays, while error bars indicate ±SD. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, non-significant.

Figure 6

Simplified model of the transcriptional regulation of three salt overly sensitive (SOS) pathway genes in response to salinity in Arabidopsis. AtbZIP62 is shown to control the expression of SOS genes in Arabidopsis plants exposed to salt stress. The loss-of-function mutant atbzip62 significantly upregulates SOS genes. AtbZIP18 and AtbZIP69 are genes coding bZIP transcription factors proposed to have an interplay with the AtbZIP62 under abiotic stress conditions. Upon salt stress perception, plants activate various metabolic and physiological processes, which include the induction of signaling components that interact with each other to provide the proper response to stress tolerance. During this event, plants redirect their resources and allocate them to protect against the stress, affecting their growth and development. ROS, reactive oxygen species. NO, nitric oxide.