The SNF1-type serine-threonine protein kinase SAPK4 regulates stress-responsive gene expression in rice

Abstract

Background: Plants respond to extracellularly perceived abiotic stresses such as low temperature, drought, and salinity by activation of complex intracellular signaling cascades that regulate acclimatory biochemical and physiological changes. Protein kinases are major signal transduction factors that have a central role in mediating acclimation to environmental changes in eukaryotic organisms. In this study, we characterized the function of the sucrose nonfermenting 1-related protein kinase2 (SnRK2) SAPK4 in the salt stress response of rice.

Results: Translational fusion of SAPK4 with the green fluorescent protein (GFP) showed subcellular localization in cytoplasm and nucleus. To examine the role of SAPK4 in salt tolerance we generated transgenic rice plants with over-expression of rice SAPK4 under control of the CaMV-35S promoter. Induced expression of SAPK4 resulted in improved germination, growth and development under salt stress both in seedlings and mature plants. In response to salt stress, the SAPK4-overexpressing rice accumulated less Na+ and Cl- and showed improved photosynthesis. SAPK4-regulated genes with functions in ion homeostasis and oxidative stress response were identified: the vacuolar H+-ATPase, the Na+/H+ antiporter NHX1, the Cl- channel OsCLC1 and a catalase.

Conclusion: Our results show that SAPK4 regulates ion homeostasis and growth and development under salinity and suggest function of SAPK4 as a regulatory factor in plant salt stress acclimation. Identification of signaling elements involved in stress adaptation in plants presents a powerful approach to identify transcriptional activators of adaptive mechanisms to environmental changes that have the potential to improve tolerance in crop plants.

Figure 1

Effect of salt stress on the transcript abundance of SAPK4 in leaves of F. rubra ssp. litoralis and rice grown under control conditions and treated with NaCl for 6 h, 24 h, and 48 h, respectively. The transcript levels of SAPK4 were quantified by semiquantitative RT-PCR. (A) RT-PCR amplification of fragments of the coding region of SAPK4. 0 – control, 125 – 125 mM NaCl, 250 – 250 mM NaCl, 500 – 500 mM NaCl. Actin was amplified as a loading control. (B) Densitometric analysis of the transcript levels of SAPK4. The transcript amounts of SAPK4 in leaves of F. rubra ssp. litoralis and rice grown under control conditions were each set to 100%. The transcript amounts were normalized to actin. Data represent means ± SD. (n = 3).

Figure 2

Subcellular localization of SAPK4-protein.(A) Nuclear localization of SAPK4-GFP fusion protein in onion epidermal cells. The arrow points to the nucleus. (B) The GFP-derived fluorescence signal of SAPK4-GFP fusion protein was merged with a light microscopic image of the transformed onion epidermal cell. (C) Onion epidermal cells transformed with a translational construct of GFP as a positive control showed localization throughout the cell with strongest signals in cytoplasm and nucleus. (D) Onion epidermal cells transformed with the empty vector as a background control. (E) Cytoplasmic localization of SAPK4-GFP fusion proteins in protoplasts of A. thaliana. nu – nucleus, ch – chloroplast, cy – cytoplasm.

Figure 3

Increased salt tolerance in transgenic rice plants over-expressing SAPK4.(A) Northern-type hybridization of the expression of SAPK4 in leaves of wild-type rice (WT) and the SAPK4 over-expressing rice lines S1 and S4 grown under control conditions and analysis of the SAPK4 transcript levels by RT-PCR in leaves of wild-type rice (WT) and the SAPK4 over-expressing rice lines S1, S4, and S5 exposed to 150 mM NaCl for 48 hours. Analysis of the SAPK4 transcript levels by RT-PCR in leaves of wild-type rice (WT) and the rice lines C1 and C2 that were transformed with the empty plant expression vector and that were exposed to 150 mM NaCl for 48 hours is shown as a control. Transcript levels of actin are shown as a loading control. (B) Phenotype of wild-type rice and transgenic rice plants over-expressing SAPK4. The plants were grown to the age of 8 weeks in hydroponic culture. Control plants and plants that were treated with 150 mM NaCl for up to 7 days. (C) Growth performance of wild-type rice and the SAPK4-over-expressing lines. Values are means ± S.D. (n = 30).

Figure 4

Increased germination efficiency and seedling development in SAPK4-over-expressing rice.(A) Germination rate of wild-type rice and the SAPK4 over-expressing rice lines S1 and S4 under control conditions and after treatment with 50 mM NaCl for 7 days. Values are means ± S.D. (n = 30). * The germination rates of wild type rice under control and under salt stress conditions are significantly different (p < 0.05). (B) Phenotype of seedlings grown under control conditions and after treatment with 50 mM NaCl for 7 days.

Figure 5

Ion accumulation and photosynthetic quantum yield ΦPSII in SAPK4-over-expressing rice in response to salt stress.(A) Reduced Cl^- content in leaves of 8-week-old SAPK4-over-expressing lines S1 and S4 treated with 150 mM NaCl for 48 h compared with wild-type rice grown under control conditions and salt stress. Values are means ± S.D. (n = 30). (B) Na⁺, K⁺ and Ca²⁺ content in leaves of wild-type rice and the SAPK4-over-expressing line S4. The plants were grown in hydroponic culture to the age of 3 weeks and were treated with 150 mM NaCl for 48 h. Values are means ± S.D. (n = 7). (C) ΦPSII was calculated from chlorophyll a fluorescence. The measurements were performed in attached leaves of 8-week-old control plants and plants treated with 150 mM NaCl for 48 h. Data represent means ± S.D. n = 30.

Figure 6

(A) Transcript accumulation of SAPK4-regulated genes in wild-type rice (WT) under control conditions and under salt stress. Transcript levels were determined by RT-PCR from total RNA isolated from 8-week-old plants. The plants were grown in hydroponic culture and stressed with 150 mM NaCl for 48 h. (B) Transcript accumulation of SAPK4-regulated genes in wild-type rice (WT) and the SAPK4-over-expressing rice lines S1 and S4. Transcript levels were determined by RT-PCR from total RNA isolated from 8-week-old plants. The plants were grown in hydroponic culture and treated with 150 mM NaCl for 48 h. Actin was amplified as a loading control.

Figure 7

Model on the putative involvement of the SNF1-type serine-threonine protein kinase SAPK4 in the regulation of gene expression in response to salinity. Catalases are involved in intracellular ROS detoxification and maintenance of photosynthesis [for example 54], the vacuolar ATPase (VHA) energizes the tonoplast NHX-type Na⁺/H⁺ antiporter for vacuolar Na⁺ sequestration [55], and transcription of voltage gated Cl^--channels is regulated salt-dependently in rice [48].