The Halophyte Halostachys caspica AP2/ERF Transcription Factor HcTOE3 Positively Regulates Freezing Tolerance in Arabidopsis

Abstract

The APETALA2 (AP2) and ethylene-responsive element-binding factor (ERF) gene family is one of the largest plant-specific transcription factor gene families, which plays a critical role in plant development and evolution, as well as response to various stresses. The TARGET OF EAT3 (TOE3) gene is derived from Halostachys caspica and belongs to the AP2 subfamily with two AP2 DNA-binding domains. Currently, AP2 family mainly plays crucial roles in plant growth and evolution, yet there are few reports about the role of AP2 in abiotic stress tolerance. Here, we report HcTOE3, a new cold-regulated transcription factor gene, which has an important contribution to freezing tolerance. The main results showed that the expression of HcTOE3 in the H. caspica assimilating branches was strongly induced by different abiotic stresses, including high salinity, drought, and extreme temperature (heat, chilling, and freezing), as well as abscisic acid and methyl viologen treatments. Overexpressing HcTOE3 gene (OE) induced transgenic Arabidopsis plant tolerance to freezing stress. Under freezing treatment, the OE lines showed lower content of malondialdehyde and electrolyte leakage and less accumulation of reactive oxygen species compared with the wild type. However, the survival rates, antioxidant enzyme activities, and contents of osmotic adjustment substance proline were enhanced in transgenic plants. Additionally, the OE lines increased freezing tolerance by up-regulating the transcription level of cold responsive genes (CBF1, CBF2, COR15, COR47, KIN1, and RD29A) and abscisic acid signal transduction pathway genes (ABI1, ABI2, ABI5, and RAB18). Our results suggested that HcTOE3 positively regulated freezing stress and has a great potential as a candidate gene to improve plant freezing tolerance.

Keywords: AP2/ERF transcription factor; Halostachys caspica; HcTOE3; freezing tolerance; transgenic Arabidopsis.

FIGURE 1

The analysis of the conserved domains (A), phylogenetic relationship (B), transcriptional activation of HcTOE3 (C), and subcellular localization of HcTOE3 (D).

FIGURE 2

Expression profile of H. caspica assimilating branch TOE3 gene under different abiotic stress and abscisic acid treatments: 600 mM NaCl (A), 1,000 mM mannitol (B), 45°C high temperature (C), 100 μM MV (D), 4°C chilling injury (E), 0°C freezing point (F), −2°C freezing stress (G), 300 μM abscisic acid (ABA) (H). HcUBC10 served as reference gene, and the expression of HcTOE3 gene in the control (0 h) was adjusted to 1. Data represented the average of three biological replicates. Significant differences at *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 3

The identification and selection of overexpression Arabidopsis. (A) Genomic DNA PCR analysis of HcTOE3 gene from WT and four transgenic lines. M, DNA marker; N, negative control using water as template. (B) Relative expression of HcTOE3 in WT and four transgenic lines with AtActin2 as an internal control using qRT-PCR. Data represented the average of three biological replicates. Significant differences were at ***p < 0.001.

FIGURE 4

Overexpression of HcTOE3 confers freezing tolerance in Arabidopsis. (A) Phenotype of the HcTOE3-overexpressing plants and the non-transgenic controls under freezing stress. (B) Survival rates were counted from 46 biological replicates. (C) Electrolyte leakage. (D) MDA content. Data represented the average of three biological replicates. Significant differences at *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 5

The performance of HcTOE3 transgenic Arabidopsis under freezing treatment. (A) H₂O₂ detection in plant leaves by DAB staining. (B) O₂^– detection in plant leaves by NBT staining. (C, D) Dead cell detection in plant leaves by Evans blue and trypan blue staining. (E) SOD activity. (F) POD activity. (G) CAT activity. (H) Proline content. (I–K) Expression levels of antioxidant enzyme genes, including POD, APX, and CAT. (L) Expression level of osmotic regulation gene P5CS. Data represented the average of three biological replicates. Significant differences at *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 6

Expression patterns of cold-responsive and ABA-associated genes in the transgenic Arabidopsis and the non-transgenic plants under freezing stress treatment. (A–F) Expression profiles of cold responsive genes, including CBF1, CBF2, COR15A, COR47, KIN1, and RD29A. (G–J) Expression profiles of ABA-related genes, including ABI1, ABI2, ABI5, and RAB18. Data represented the average of three biological replicates. Significant differences at *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 7

Model of HcTOE3 regulation of the freezing stress response in transgenic Arabidopsis. The model indicated that overexpression of HcTOE3 up-regulates genes taking part in CBFs-COR signaling, ABA signaling, proline biosynthesis, and ROS scavenging. These genes lead to an increase in SOD, POD, CAT activities and proline content; a reduction ROS content and cell membrane peroxidation; and ultimately enhancing the freezing tolerance of Arabidopsis.