Transcription Factor CaSBP12 Negatively Regulates Salt Stress Tolerance in Pepper (Capsicum annuum L.)

Abstract

SBP-box (Squamosa-promoter binding protein) genes are a type of plant-specific transcription factor and play important roles in plant growth, signal transduction, and stress response. However, little is known about the role of pepper SBP-box transcription factor genes in response to abiotic stress. Here, one of the pepper SBP-box gene, CaSBP12, was selected and isolated from pepper genome database in our previous study. The CaSBP12 gene was induced under salt stress. Silencing the CaSBP12 gene enhanced pepper plant tolerance to salt stress. The accumulation of reactive oxygen species (ROS) of the detached leaves of CaSBP12-silenced plants was significantly lower than that of control plants. Besides, the Na⁺, malondialdehyde content, and conductivity were significantly increased in control plants than that in the CaSBP12-silenced plants. In addition, the CaSBP12 over-expressed Nicotiana benthamiana plants were more susceptible to salt stress with higher damage severity index percentage and accumulation of ROS as compared to the wild-type. These results indicated that CaSBP12 negatively regulates salt stress tolerance in pepper may relate to ROS signaling cascades.

Keywords: CaSBP12; Nicotiana benthamiana; pepper; reactive oxygen species; salt stress.

Figure 1

Phenotypes and silencing efficiency of CaSBP12 in silenced and control plants. (A) The phenotypes of CaSBP12-silenced plants. Photographs were taken 40 days post-infiltration. The white line is used as a scale bar (length 3.5 cm) (the diameter of the pot is 7 cm). (B) Silencing efficiency of CaSBP12 in CaSBP12-silenced plants. ** Represents significant differences at p ≤ 0.01. Mean values and SDs for three biological replicates are shown.

Figure 2

Phenotypes of the detached leaves of CaSBP12-silenced and control plants after 2 days of salt stress. (A) Phenotypes of the detached leaves of silenced plants after 2 days of treatment with water. (B) Phenotypes of the detached leaves of silenced plants after 2 days of treatment with 200 mM NaCl. (C) Phenotypes of the detached leaves of silenced plants after 2 days of treatment with 400 mM NaCl. (D) Phenotypes of the detached leaves of silenced plants after 2 days of treatment with 600 mM NaCl. (E) Water-stained area (%) of the detached leaves of silenced plants after 2 days of treatment with salt stress. (F) The total chlorophyll content of the detached leaves of silenced plants after 2 days of treatment with salt stress. (G) The net photosynthetic rate (Pn) of the detached leaves of silenced plants after 2 days of treatment with salt stress. (H) The Na⁺ content of the detached leaves of silenced plants after 2 days of treatment with salt stress. The black line used as a scale bar (length 0.5 cm). * and ** represent significant differences at p ≤ 0.05 and p ≤ 0.01 respectively. Mean values and SDs at least for three biological replicates are shown.

Figure 3

DAB and NBT staining of silenced plants and the phenotypes of the detached leaves of silenced plants. (A) Phenotypes of the detached leaves of CaSBP12-silenced and control plants after 2 days of treatment with 400 mM NaCl. (B) DAB staining in leaves of CaSBP12-silenced and control plants after 2 days of treatment with 400 mM NaCl. (C) NBT staining in leaves of CaSBP12-silenced and control plants after 2 days of treatment with 400 mM NaCl. (D) Water-stained area (%) of the detached leaves of silenced plants after 2 days of treatment with 400 mM NaCl. (E) The DAB stained area (%) of the detached leaves of silenced plants after 2 days of treatment with 400 mM NaCl. (F) The NBT stained area (%) of the detached leaves of silenced plants after 2 days of treatment with 400 mM NaCl. (G) The H₂O₂ content of the detached leaves of silenced plants after 2 days of treatment with 400 mM NaCl. d: days. The black line is used as a scale bar (length 0.75 cm). Bars with different letters indicate significant differences at p ≤ 0.05. Mean values and SDs at least for three biological replicates are shown.

Figure 4

Phenotypes, total chlorophyll, Na⁺, malondialdehyde (MDA) content and conductivity of silenced plants after 3 days of salt stress. (A) Phenotypes of CaSBP12-silenced and control plants after 3 days of treatment with 400 mM NaCl. The red line is used as a scale bar (length 2 cm). (B) The total chlorophyll content of CaSBP12-silenced and control plants after 3 days of treatment with 400 mM NaCl. (C) The Na⁺, MDA content, and conductivity of CaSBP12-silenced and control plants after 3 days of treatment with 400 mM NaCl. d: days. Bars with different letters indicate significant differences at p ≤ 0.05. Mean values and SDs for three biological replicates are shown.

Figure 5

The expression of reactive oxygen species (ROS)-scavenging enzymes related genes after 3 days of salt stress (400 mM NaCl) in silenced plants. Bars with different letters indicate significant differences at p ≤ 0.05. Mean values and SDs for three biological replicates are shown.

Figure 6

Overexpression of CaSBP12 in Nicotiana Benthamiana enhanced susceptibility to salt stress. (A) Phenotypes of transgenic (Line 4, Line 7 and Line 8) and wild-type lines after 22 days of treatment with 200 mM NaCl. (B) The total chlorophyll content of transgenic and wild-type lines after 22 days of treatment with 200 mM NaCl. (C) The relative water content of transgenic and wild-type lines during treatment with 200 mM NaCl at day 0, day 7, day 14, and day 22. (D) The Na⁺ content of transgenic and wild-type lines after 22 days of treatment with 200 mM NaCl. (E) The MDA content of transgenic and wild-type lines after 22 days of treatment with 200 mM NaCl. (F) The conductivity of transgenic and wild-type lines after 22 days of treatment with 200 mM NaCl. (G) Classification of damage severity index percentage of transgenic and wild-type lines after 22 days of treatment with 200 mM NaCl. Level 0: no symptoms; Level 1: yellowing of lower leaves of plants; Level 2: the whole plant is yellowing and the edge of leaves is decolorized seriously; Level 3: whole plant yellowing and growth point death. (H) Damage severity index percentage of transgenic and wild-type lines after 22 days of treatment with 200 mM NaCl. The white line used as a scale bar (length 3.5 cm). Bars with different letters indicate significant differences at p ≤ 0.05. Mean values and SDs for three replicates are shown.

Figure 7

DAB and NBT staining of transgenic and wild-type lines after 22 days of salt stress. (A) DAB staining of transgenic and wild-type lines after 22 days of treatment with 200 mM NaCl. (B) NBT staining of transgenic and wild-type lines after 22 days of treatment with 200 mM NaCl. (C) The DAB, NBT stained area (%) and H₂O₂ content of transgenic and wild-type lines after 22 days of treatment with 200 mM NaCl. The black line is used as a scale bar (length 0.75 cm). * and ** represent significant differences at p ≤ 0.05 and p ≤ 0.01 respectively. Mean values and SDs at least for three biological replicates are shown.

Figure 8

The expression of ROS-scavenging enzymes related genes in transgenic and wild-type lines after 22 days of salt stress. Bars with different letters indicate significant differences at p ≤ 0.05. Mean values and SDs for three biological replicates are shown.