Overexpression of a Fragaria vesca MYB Transcription Factor Gene (FvMYB82) Increases Salt and Cold Tolerance in 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 to different abiotic stresses, such as salt, cold, and drought. In the present study, a new MYB TF gene was cloned from Fragaria vesca (a diploid strawberry) and named FvMYB82. The open reading frame (ORF) of FvMYB82 was found to be 960 bp, encoding 319 amino acids. Sequence alignment results and predictions of the protein structure indicated that the FvMYB82 contained the conserved R2R3-MYB domain. Subcellular localization analysis showed that FvMYB82 was localized onto the nucleus. Furthermore, the qPCR showed that the expression level of FvMYB82 was higher in new leaves and roots than in mature leaves and stems. When dealing with different stresses, the expression level of FvMYB82 in F. vesca seedlings changed markedly, especially for salt and cold stress. When FvMYB82 was introduced into Arabidopsis thaliana, the tolerances to salt and cold stress of FvMYB82-OE A. thaliana were greatly improved. When dealt with salt and cold treatments, compared with wild-type and unloaded line (UL) A. thaliana, the transgenic lines had higher contents of proline and chlorophyll, as well as higher activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). However, the transgenic A. thaliana had lower level of malondialdehyde (MDA) and electrolytic leakage (EL) than wild-type and UL A. thaliana under salt and cold stress. Meanwhile, FvMYB82 can also regulate the expression of downstream genes associated with salt stress (AtSnRK2.4, AtSnRK2.6, AtKUP6, and AtNCED3) and cold stress (AtCBF1, AtCBF2, AtCOR15a, and AtCOR78). Therefore, these results indicated that FvMYB82 probably plays an important role in the response to salt and cold stresses in A. thaliana by regulating downstream related genes.

Keywords: Fragaria vesca; FvMYB82; abiotic stress; low-temperature stress; salt stress.

Figure 1

Contrast and evolutionary relationship between FvMYB82 and MYB transcription factors of different varieties. (A) Comparison between homology of FvMYB82 protein and MYB protein in other plants. The conserved regions of the amino acid sequence are marked by black and red boxes. (B) Phylogenetic tree analysis of MYB protein in FvMYB82 and other plants. The accession numbers are as follows: RcMYB82 (Rosa chinensis, XP_024181819.1), MdMYB82 (Malus domestica, RXI05710.1), PuMYB82 (Pyrus ussuriensis, KAB2620502.1), PbMYB82 (Pyrus bretschneideri, XP_009363992.1), MbMYB82 (Malus baccata, TQE02283.1), JrMYB82 (Juglans regia, XP_018814279.1), ZjMYB82 (Ziziphus jujuba, XP_015867230.1), QlMYB82 (Quercus lobata, XP_030931205.1), CiMYB82 (Carya illinoinensis, KAG6718834.1), RrMYB82 (Rhamnella rubrinervis, KAL3453734.1), ToMYB82 (Trema orientale, PON99826.1), GhMYB82 (Gossypium hirsutum, XP_016720794.1), JmMYB82 (Juglans microcarpa, XP_041006667.1).

Figure 2

Prediction of FvMYB82 protein domains and structure. (A) Predicted protein secondary structure; (B) predicted protein domains; (C) predicted tertiary structure.

Figure 3

Subcellular localization of FvMYB82 in onion leaf epidermal cells. The 35S:GFP and 35S:FvMYB82 plasmids were transformed into the cells by particle bombardment. (A,D) Bright-field images, (B,E) GFP fluorescence, (C,F) cells stained with DAPI. Bar = 50 μm.

Figure 4

Expression pattern analysis of FvMYB82 in F. vesca by quantitative RT-PCR. (A) Expression of FvMYB82 in different tissues in the non-stress environment. (B,C) Time course of FvMYB82 expression in young and root in the control and under treatment with salt (200 mM NaCl), heat (30 °C), cold (4 °C), dehydration (15% PEG6000), and abscisic acid (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

Growth of transgenic A. thaliana lines overexpressing FvMYB82 under salt treatment. (A) Relative expression level of FvMYB82 in WT, UL and 5 FvMYB82-overexpression lines (L1, L2, L3, L4 and L5). (B) Phenotypes of the WT, UL, and transgenic lines (L1, L3, and L5) grown in the control environment, salt treatment (irrigation with 200 mM NaCl for 7 days), and recovery after salt treatment (irrigation with water for 3 days). Bar = 5 cm. (C) Survival percentages of WT, UL, and transformed lines (L1, L3, and L5). Asterisks indicate significant differences between WT and UL, transformed lines (** p ≤ 0.01).

Figure 6

Physiological indicators in transgenic A. thaliana lines overexpressing FvMYB82 under salt treatment. Contents of (A) Chlorophyll, (B) MDA, (C) proline, and the activities of (D) CAT, (E) SOD, and (F) POD in the WT, UL, and FvMYB82-OE lines (L1, L3, and L5) under 200 mM NaCl treatment for 7 days. Significant differences were marked with asterisks above the error bar (* p ≤ 0.05, ** p ≤ 0.01). The levels of indicators in the WT were used as the control.

Figure 7

Expression levels of salt-related genes in WT, UL, and transformed A. thaliana. overexpressing FvMYB82 under salt stress. Relative expression levels of (A) AtSnRK2.4, (B) AtSnRK2.6, (C) AtKUP6, and (D) AtNCED3 in the WT, UL, and FvMYB82-OE lines (L1, L3, and L5). Data are the average of three replicates. Significant differences are marked with an asterisk above the error bar (** p ≤ 0.01).

Figure 8

Growth of transgenic A. thaliana overexpressing FvMYB82 under low-temperature treatment. (A) Phenotypes of WT, transformants with empty vector (UL), and FvMYB82-overexpressing lines (L1, L3, and L5) under the control environment (22 °C), cold treatment (4 °C), and after recovery. Bar = 1 cm. (B) Survival rate of WT, UL, and transgenic lines under the control environment and cold treatment. Three replicates were performed. Asterisks indicate a significant difference between the different lines (** p ≤ 0.01).

Figure 9

Physiological indicators in transgenic A. thaliana lines overexpressing FvMYB82 under low-temperature treatment. (A) Proline content, (B) MDA content, (C) chlorophyll content, (D) CAT activity, (E) SOD activity, and (F) POD activity in the WT, UL, and FvMYB82-overexpressing lines (L1, L3, and L5) under the non-stress environment (22 °C) or cold treatment (4 °C for 12 h). Asterisks above each error bar indicate obviously significant differences between transgenic lines (L1, L3, and L5), UL and the WT (* p ≤ 0.05, ** p ≤ 0.01). The levels of indicators in the WT were used as the control.

Figure 10

Expression of chilling-related genes in transgenic A. thaliana lines overexpressing FvMYB82 under low-temperature treatment. Relative expression levels of (A) ATCBF1, (B) AtCBF2, (C) AtCOR15a, and (D) AtCOR78 in the WT, UL, and transgenic lines (L1, L3, and L5). Data are the average of three repetitions. Asterisks indicate extremely significant differences between the transgenic line and the WT (** p ≤ 0.01).

Figure 11

Model of FvMYB82 response to low temperature and salt stress. Salt stress promotes expression of FvMYB82, increases the ABA biosynthesis and expression level of signal transduction genes SnRK2.4 SnRK2.6, and improves salt tolerance. Subsequently, ABA production stimulates the expression of FvMYB82. In addition, salt stress induces the expression of the crucial salt-responsive genes AtKUP6 and AtNCED3 and increases plant salt tolerance. Cold stress induces the expression of FvMYB82, and FvMYB82 domain binds to the promoter regions of CBF1 and CBF2 and promotes binding of CBFs to the CRT/DRE cis-acting elements of downstream genes, thereby activating expression of the downstream cold-responsive genes COR15a and COR78. It also enhances plant cold tolerance.