Molecular Cloning and Characterization of MbMYB108, a Malus baccata MYB Transcription Factor Gene, with Functions in Tolerance to Cold and Drought Stress in Transgenic Arabidopsis thaliana

Abstract

The MYB transcription factor (TF) family is one of the largest transcription families in plants, which is widely involved in the responses of plants to biotic and abiotic stresses, as well as plant growth, development, and metabolic regulation. In the present study, a new MYB TF gene, MbMYB108, from Malus baccata (L.) Borkh, was identified and characterized. The open reading frame (ORF) of MbMYB108 was found to be 903 bp, encoding 300 amino acids. Sequence alignment results and predictions of the protein structure indicated that the MbMYB108 protein contained the conserved MYB domain. Subcellular localization showed that MbMYB108 was localized to the nucleus. The expression of MbMYB108 was enriched in young and mature leaves, and was highly affected by cold and drought treatments in M. baccata seedlings. When MbMYB108 was introduced into Arabidopsis thaliana, it greatly increased the cold and drought tolerances in the transgenic plant. Increased expression of MbMYB108 in transgenic A. thaliana also resulted in higher activities of peroxidase (POD) and catalase (CAT), higher contents of proline and chlorophyll, while malondialdehyde (MDA) content and relative conductivity were lower, especially in response to cold and drought stresses. Therefore, these results suggest that MbMYB108 probably plays an important role in the response to cold and drought stresses in A. thaliana by enhancing the scavenging capability for reactive oxygen species (ROS).

Keywords: Malus baccata (L.) Borkh; MbMYB108; cold stress; drought stress.

Figure 1

Comparison and phylogenetic relationship of MbMYB108 with other species of MYB transcription factor (TF) proteins. (A) Amino acid sequence alignment of MbMYB108 with other species of MYB TF proteins. Conserved domains are shown in red and blue boxes. (B) Phylogenetic tree analysis of MbMYB108 and other species of MYB TF proteins.

Figure 2

Structural prediction of MbMYB108 protein. Prediction of the secondary structure (A), domains (B), and tertiary structure (C).

Figure 3

Subcellular localization of MbMYB108 protein. Transient expressions of green fluorescent protein (GFP) and MbMYB108-GFP fusion protein in onion epidermal cells was observed by fluorescence microscopy: (A,D) were taken under bright light, (B,E) were taken under dark field, and (C,F) are the results of DAPI staining for 24 h. Scale bar corresponds to 5 μm.

Figure 4

Time-course expression patterns of MbMYB108 in Malus baccata. (A) Expression patterns of MbMYB108 in young leaves (partly expanded), mature leaves (fully expanded), roots, and stems in normal condition (room temperature and normal watering). The expression level of young leaves was used as a control. Expression patterns of MbMYB108 in control condition (CK), low-temperature (4 °C), high-salt (200 mM NaCl), dehydration (20% PEG), high-temperature (38 °C), and ABA (50 μM) stress in young leaves (B) and roots (C) at the following time points: 0, 1, 3, 5, 7, 9, and 12 h. Data represent the mean of three replicates. Error bars represent standard deviation. Asterisks above error bars indicate significant differences between treatment and control (0 h) (* p ≤ 0.05, ** p ≤ 0.01).

Figure 5

Overexpression of MbMYB108 in Arabidopsis thaliana improved cold tolerance. (A) Transgenic A. thaliana qPCR validation. (B) Phenotypic map of each line (WT, UL, S2, S4, and S6) of A. thaliana under CK (Cold 0 h), cold stress (Cold 14 h), and recovery conditions (stress removed). Scale bar corresponds to 4 cm. (C) The survival rates of each line (WT, UL, S2, S4, and S6) of A. thaliana under CK and cold stress. (D) Catalase (CAT) activity. (E) Chlorophyll content. (F) Peroxidase (POD) activity. (G) Proline content. (H) Malondialdehyde (MDA) content. (I) Relative conductivity. Asterisks above the error bars indicate extremely significant differences between transgenic (S2, S4, S6) and WT A. thaliana (** p ≤ 0.01). The level of each index in the WT line was used as a control.

Figure 6

Relative expression levels of cold stress-related genes in WT, UL, and transgenic A. thaliana. Relative expression levels of AtCBF1 (A), AtCBF3 (B), AtCOR15a (C), and AtRD29a (D). Data represent the mean of three replicates. Error bars represent standard deviation. Asterisks above error bars indicate significant difference compared to the WT line (** p ≤ 0.01).

Figure 7

Overexpression of MbMYB108 in A. thaliana improved drought tolerance. (A) Phenotypic map of each line (WT, UL, S2, S4, and S6) of A. thaliana under CK (Drought 0 days), drought stress (Drought 10 days), and recovery conditions (stress removed). Scale bar corresponds to 4 cm. (B) The survival rates of each line (WT, UL, S2, S4, and S6) of A. thaliana under CK and drought stress. (C) Catalase (CAT) activity. (D) Chlorophyll content. (E) Peroxidase (POD) activity. (F) Proline content. (G) Malondialdehyde (MDA) content. (H) Relative conductivity. Asterisks above the error bars indicate extremely significant differences between transgenic (S2, S4, S6) and WT A. thaliana (** p ≤ 0.01). The level of each index in the WT line was used as a control.

Figure 8

Relative expression levels of drought stress-related genes in WT, UL, and transgenic A. thaliana. Relative expression levels of AtNCED3 (A), AtSnRK2.4 (B), AtCAT1 (C), and AtP5CS (D). Data represent the mean of three replicates. Error bars represent standard deviation. Asterisks above error bars indicate significant difference compared to the WT line (** p ≤ 0.01).