The Wheat MYB Transcription Factor TaMYB31 Is Involved in Drought Stress Responses in Arabidopsis

Abstract

Drought is one of the major environmental stresses limiting crop growth and production. MYB family transcription factors play crucial roles in response to abiotic stresses. Previous studies found that TaMYB31 is transcriptionally induced by drought stress. However, the biological functions of TaMYB31 in drought stress responses remained unknown. In this study, three TaMYB31 homoeologous genes from hexaploid wheat, designated TaMYB31-A, TaMYB31-B, and TaMYB31-D, were cloned and characterized. Expression analysis showed that TaMYB31 genes have different tissue expression patterns, and TaMYB31-B has relatively high expression levels in most tested tissues. All the three homoeologs were up-regulated by polyethylene glycol (PEG) 6000 and abscisic acid (ABA) treatments. Subcellular localization analyses revealed that TaMYB31 is localized to the nucleus. Ectopic expression of the TaMYB31-B gene in Arabidopsis affected plants growth and enhanced drought tolerance. In addition, seed germination and seedling root growth of TaMYB31-B transgenic plants were more sensitive to exogenous ABA treatment compared to wild type control. RNA-seq analysis indicated that TaMYB31 functions through up-regulation of wax biosynthesis genes and drought-responsive genes. These results provide evidence that TaMYB31 acts as a positive regulator of drought resistance, and justify its potential application in genetic modification of crop drought tolerance.

Keywords: Arabidopsis; MYB; RNA-seq; drought; transgenic; wheat.

Figure 1

Sequence analysis of TaMYB31 homologs. (A) Comparison of amino acid sequences of seven MYB31 proteins, i.e., hexaploid wheat TaMYB31-A/B/D (GenBank: MH428951-53), Triticum urartu TuMYB31 (GenBank: MH428954), Aegilops speltoides AsMYB31 (GenBank: MH428955), Aegilops tauschii AetMYB31 (GenBank: MH428956), and AtMYB96 (AT5G62470). The SANT domain is underlined. (B) Phylogenetic and gene structure analysis of TaMYB31 homoeologs, three diploid ancestral wheat MYB31 genes and AtMYB96. Exons and introns are represented by black boxes and lines, respectively.

Figure 2

Expression patterns of TaMYB31s. (A) TaMYB31s expression in various wheat tissues by quantitative RT-PCR. YS, young spikes and FS, flowering spikes. Data are presented as means of three replicates ± SD. (B,C) Expression of TaMYB31s under PEG (B) and ABA treatment (C) for the indicated time points by quantitative RT-PCR. Data are presented as means of three replicates ± SD.

Figure 3

Subcellular localization of TaMYB31-B in Nicotiana benthamiana leaf epidermal cells. 35S::GFP and 35S::TaMYB31-B-GFP plasmids were transformed into Nicotiana benthamiana leaf epidermal cells and GFP fluorescence were visualized under a confocal laser-scanning microscope. From left to right, GFP fluorescence (A,D), bright-field (B,E), and overlays of the GFP fluorescence and bright-field (C,F). Bar = 40 μm.

Figure 4

Generation of transgenic Arabidopsis plants overexpressing TaMYB31-B. (A) Schematic diagram of the 35S::TaMYB31-B construct. LB left border, p35S cauliflower mosaic virus 35S promoter, Tnos nopaline synthase gene (NOS) terminator, Bar bialaphos-resistance gene, RB right border. (B) Transcript levels of TaMYB31-B in the leaves of T₃ transgenic Arabidopsis lines via quantitative RT-PCR analysis. Bars indicate standard deviations of three replicates. WT non-transgenic plants, N.D, Not Detected. (C) Phenotypes of TaMYB31-B transgenic plants. Bar = 2 cM. (D) Statistical analyses of rosette diameter, plant height and fresh weight of mature plants grown in soil. ^* and ^** indicate significant differences at P < 0.05 and P < 0.01 levels, respectively, by Student's t-test.

Figure 5

Drought tolerance responses of TaMYB31-B overexpressing transgenic lines. (A) Images of drought tolerance assays and (B) survival rates of WT and TaMYB31-B overexpressing lines of the T₃ generation. Watering was withheld for two weeks and re-applied for 3 days. (C) Water loss assays in detached leaves of WT and transgenic lines. ^**Indicates significant differences at P < 0.01 level by Student's t-test.

Figure 6

Phenotypes of WT and TaMYB31-B transgenic plants under PEG stress. (A) Images of WT and transgenic lines under PEG treatment. (B) Fresh weight and root lengths of Arabidopsis seedlings. Data are presented as means ± SD of three replicates (^*P < 0.05, ^**P < 0.01).

Figure 7

Seed germination rates analysis of WT and TaMYB31-B transgenic lines under ABA treatment. (A) Seeds of WT and three T3 TaMYB31-B transgenic lines grown on 1/2MS agar plates with the indicated concentrations of ABA. The photographs were taken 4 d after sowing. (B) Seed germination rates recorded before photographing. (C) Morphology of WT and transgenic seedlings on plates without or with 10 μM ABA. Images were taken 3 d after seedlings were transferred to the shown plates. (D) Root elongation rates of WT and transgenic seedlings at 3 d after being transferred to the plates without or with 10 μM ABA. Error bars indicate SD of three independent experiments (^*P < 0.05, ^**P < 0.01).

Figure 8

Transcriptome analysis of the 35S::TaMYB31-B transgenic Arabidopsis under drought conditions. (A) Number of up- or down-regulated genes in transgenic plants compared with WT plants using a significant cutoff of false discovery rate (FDR) < 0.01, and a fold change (FC) > 2. (B) Gene ontology classification for differentially expressed genes (DEGs) between WT and TaMYB31-B transgenic plants. (C) Confirmation of DEGs expression by quantitative RT-PCR analysis. Error bars represent SD of three replicates.