Genome wide analysis of the apple MYB transcription factor family allows the identification of MdoMYB121 gene confering abiotic stress tolerance in plants

Abstract

The MYB proteins comprise one of the largest families of transcription factors (TFs) in plants. Although several MYB genes have been characterized to play roles in secondary metabolism, the MYB family has not yet been identified in apple. In this study, 229 apple MYB genes were identified through a genome-wide analysis and divided into 45 subgroups. A computational analysis was conducted using the apple genomic database to yield a complete overview of the MYB family, including the intron-exon organizations, the sequence features of the MYB DNA-binding domains, the carboxy-terminal motifs, and the chromosomal locations. Subsequently, the expression of 18 MYB genes, including 12 were chosen from stress-related subgroups, while another 6 ones from other subgroups, in response to various abiotic stresses was examined. It was found that several of these MYB genes, particularly MdoMYB121, were induced by multiple stresses. The MdoMYB121 was then further functionally characterized. Its predicted protein was found to be localized in the nucleus. A transgenic analysis indicated that the overexpression of the MdoMYB121 gene remarkably enhanced the tolerance to high salinity, drought, and cold stresses in transgenic tomato and apple plants. Our results indicate that the MYB genes are highly conserved in plant species and that MdoMYB121 can be used as a target gene in genetic engineering approaches to improve the tolerance of plants to multiple abiotic stresses.

Figure 1. The DNA-binding domains of MdR2R3 MYB proteins and chromosomal locations for MdoMYB genes.

The sequence logs of the R2 (A) and R3 (B) MYB repeats are generated by submitting the multiple alignments of all MdR2R3 MYB domains with ClustalX to the website of WebLogo software, which is used to analysis conserved residues (http://weblogo.berkeley.edu/logo.cgi). The overall height of each stack indicates the conservation of the sequence at that position and the bit score indicates the relative frequency of the corresponding acid. The conserved tryptophan residues (Trp) in the MYB domain are marked with red asterisks. The replaced residues in the R3 repeat are shown by yellow asterisks. (C) The chromosomal position of each MdoMYB gene was mapped according to the apple genome. The chromosome number is indicated at the top of each chromosome. The scale is in megabases (Mb). Blue bars on the chromosomes and red numbers inside the blue bars indicate the predicted 15 duplication regions.

Figure 2. Expression analysis of 18 MdR2R3 MYB genes under abiotic stress treatments.

The expression pattern of 18 MdR2R3 MYB genes in response to NaCl, ABA, PEG, and cold treatments at 2 h, 12 were chosen from stress-related subgroups (A) and 6 ones from other subgroups (B). MdACTIN was used as an internal standard.

Figure 3. MdoMYB121 transgenic tomato seed germination and seedling development is more tolerant to NaCl.

(A) Expression levels of MdoMYB121 in independent transgenic tomato. MdACTIN was used as an internal standard. (B) Seed germination on MS or MS supplemented with 50 mM NaCl of transgenic MdoMYB121 and the WT. All tests were repeated at least three times, and approximately 50 seeds were counted for each experiment. Data are expressed as the means ± SE. (C-D) WT and transgenic MdoMYB121 plants were germinated on MS medium and then transferred to a new MS medium supplemented with different concentrations of NaCl for 15 days.

Figure 4. Abiotic stress tolerance of MdoMYB121 transgenic tomato plants.

(A–D) Tolerance of transgenic tomato plants under normal conditions, 300 mM NaCl for 20 d, dehydration for 15 d with a 3 d recovery, 4°C for 13 d with a 3 d recovery.

Figure 5. Physiological index of tomato plants under abiotic stresses.

(A–B) Na⁺ content and Na⁺/K⁺ ratio under 300 mM salt stress for 20 d and relative water content of leaves under dehydration for 15 d of WT and MdoMYB121 transgenic tomato plants. (C) Relative electrolyte leakage, MDA and proline content under abiotic stresses of WT and MdoMYB121 transgenic tomato plants. Data are expressed as the means ± SE.

Figure 6. MdoMYB121 overexpression enhances tolerance to abiotic stresses in apple plantlets.

(A) Expression levels of MdoMYB121 in independent transgenic apple lines. (B–D) Tolerance of transgenic rooting apple plantlets for 300 mM NaCl for 16 d, dehydration for 15 d with a 3 d recovery and 4°C for 16 d with a 25 d recovery.