Overexpression of a Malus baccata (L.) Borkh WRKY Factor Gene MbWRKY33 Increased High Salinity Stress Tolerance in Arabidopsis thaliana

Abstract

The WRKY transcription factor family is a significant family of plant transcription factors (TFs). Plant growth and development are often influenced by abiotic factors, such as salinity and low temperature. Numerous studies have demonstrated that WRKY TFs primarily influence plant responses to adversity. However, there are few studies on the role of WRKY genes in the stress responses of Malus baccata (L.) Borkh. We cloned the MbWRKY33 gene from Malus baccata for this research, and its roles in salt stress tolerance were analyzed. Phylogenetic tree analysis revealed that MbWRKY33 and PbWRKY33 have the highest homology. Subcellular localization revealed that MbWRKY33 was located within the nucleus. An analysis of tissue-specific expression showed that MbWRKY33 had relatively high expression levels in young leaves and roots. Moreover, Arabidopsis thaliana plants overexpressing MbWRKY33 exhibited stronger resistance to salt stress compared with the wild type (WT) and the unloaded line empty vector (UL). Under the treatment of 200 mM NaCl, transgenic Arabidopsis thaliana plants exhibited significantly higher activities of antioxidant enzymes like superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) than the control. In contrast, the WT and the UL lines had elevated levels of malondialdehyde (MDA) and reactive oxygen species (ROS). In addition, MbWRKY33 elevates transgenic plant resistance to salt stress by regulating the expression levels of AtNHX1, AtSOS1, AtSOS3, AtNCED3, AtSnRK2, and AtRD29a. Results indicated that MbWRKY33 in Malus might be linked to high-salinity stress responses, laying a foundation for understanding WRKY TFs' reaction to such stress.

Keywords: Malus baccata; MbWRKY33; genetic transformation; high-salinity stress; transcriptional regulation.

Figure 1

Alignment and phylogenetic tree analysis of other species’ MbWRKY33 and WRKY amino acid sequence. (A) MbWRKY33 in comparison to other species’ WRKY33 proteins. Red boxes indicate two WRKY conserved structural domains; the black triangle marks the acetylene zinc finger motif of WRKY. (B) WRKY33 and MbWRKY33 protein phylogenetic trees of different species. The red underline shows the protein MbWRKY33. The numerical values represent the genetic relatedness among other species, and the phylogenetic tree was created on MEGA7.0.

Figure 2

Secondary structure and tertiary structure prediction of MbWRKY33 protein. (A) MbWRKY33’s structural analysis at the secondary level. (B) Conserved structural domain study of MbWRKY33. (C) Tertiary structural prediction of MbWRKY33.

Figure 3

Subcellular localization of MbWRKY33 protein. 35S::MbWRKY33::GFP was expressed transiently into tobacco leaves with 35Spro::GFP as a positive control. (A,E) GFP signals; (B,F) mCherry; (C,G) Bright field; (D,H) Merge. mCherry as a nuclear marker. Yellow indicates GFP and mCherry colocalization. Scale bars correspond to 20 µm.

Figure 4

Tissue-specific and stress-responsive expression patterns of MbWRKY33 in Malus baccata. (A) New leaves, stems, roots, and mature leaves all exhibit varying degrees of MbWRKY33 expression. (B) Time-course of MbWRKY33 expression in new leaves in control and under salt (200 mM NaCI), low-temperature (4 °C), dehydration (6% PEG6000), and abscisic acid (50 µM ABA) treatments. (C) Time-course of MbWRKY33 expression in roots in control and under salt (200 mM NaCl), low-temperature (4 °C), dehydration (6% PEG6000), and abscisic acid treatments (50 µM ABA). Error bars indicate the standard deviation. Asterisks above the error bars indicate a significant difference between the treatment and control (Student’s t-test; * p ≤ 0.05, ** p ≤ 0.01).

Figure 5

MbWRKY33 confers salt tolerance in Arabidopsis. (A) Expression of the MbWRKY33 transcript in transgenic lines S1–S6, null-loaded line UL, and wild-type WT. (B) Transgenic high-expression lines (S1, S3, and S4), UL, and WT phenotypes. Scale bar is 3 cm. (C) Survival of each Arabidopsis line under high-salt stress. The asterisks above indicate statistically significant comparisons (* p ≤ 0.05; ** p < 0.01) with WT differences.

Figure 6

Effects of MbWRKY33 on stress-related physiological indices in Arabidopsis under high-salt stress. Contents of (A) Relative conductivity, (B) H₂O₂, and (C) O₂⁻, (D) MDA, (E) chlorophyll, (F) proline, and the activities of (G) SOD, (H) CAT, and (I) POD in the WT, UL, and MbWRKY33-OE lines (S1, S3, and S4) under 200 mM NaCl treatment for 7 days. The mean of three replicates is the standard error. The transgenic lines differed significantly from the WT lines, as indicated by the asterisk above the error bars (Student’s t-test, ** p ≤ 0.01).

Figure 7

Effects of MbWRKY33 on the expression of genes associated with salt tolerance in Arabidopsis under high-salt stress. (A) AtNHX1; (B) AtNCED3; (C) AtSOS1; (D) AtSnRK2; (E) AtSOS3; and (F) AtRD29a. The mean of three duplicate experiments is the standard error. The transgenic lines differed significantly from the WT lines, as indicated by the asterisk above the error bars (Student’s t-test, ** p ≤ 0.01).

Figure 8

A possible model for the role of MbWRKY33 during plant resistance to salt stress. The activated MbWRKY33 substantially reduced cellular damage by reactive oxygen species and lipid peroxides, promoting the upregulated expression of stress-related genes. MbWRKY33 receives signals to be activated when plants are exposed to a high-salt environment. On the one hand, MbWRKY33 participates in the SOS pathway and binds to the promoters of AtSOS3 to activate the expression of the downstream genes, AtSOS1 and AtNHX1, to enhance the plant’s tolerance to the high-salt environment. On the other hand, MbWRKY33 participates in the ABA-signaling pathway and promotes the expression of the downstream genes AtRD29a, AtNCED3, and AtSnRK2 to realize the enhancement in salt tolerance in plants.