Overexpression of Lolium multiflorum LmMYB1 Enhances Drought Tolerance in Transgenic Arabidopsis

Abstract

Lolium multiflorum is one of the world-famous forage grasses with rich biomass, fast growth rate and good nutritional quality. However, its growth and forage yield are often affected by drought, which is a major natural disaster all over the world. MYB transcription factors have some specific roles in response to drought stress, such as regulation of stomatal development and density, control of cell wall and root development. However, the biological function of MYB in L. multiflorum remains unclear. Previously, we elucidated the role of LmMYB1 in enhancing osmotic stress resistance in Saccharomyces cerevisiae. Here, this study elucidates the biological function of LmMYB1 in enhancing plant drought tolerance through an ABA-dependent pathway involving the regulation of cell wall development and stomatal density. After drought stress and ABA stress, the expression of LmMYB1 in L. multiflorum was significantly increased. Overexpression of LmMYB1 increased the survival rate of Arabidopsis thaliana under drought stress. Under drought conditions, expression levels of drought-responsive genes such as AtRD22, AtRAB and AtAREB were up-regulated in OE compared with those in WT. Further observation showed that the stomatal density of OE was reduced, which was associated with the up-regulated expression of cell wall-related pathway genes in the RNA-Seq results. In conclusion, this study confirmed the biological function of LmMYB1 in improving drought tolerance by mediating cell wall development through the ABA-dependent pathway and thereby affecting stomatal density.

Keywords: LmMYB1; Lolium multiflorum; drought; stomatal density.

Figure 1

Multi-sequence alignment and evolutionary relationship analysis of the LmMYB1 protein. (A) Secondary structure of the LmMYB1 protein. The green boxes represent conserved domains, and the red triangles represent conserved amino acids. (B) Comparison between the homology of the LmMYB1 protein and the MYB protein in other plants. The conserved regions of the amino acid sequence are marked with green boxes. R represents the conserved domain of MYB (W-X₁₉-W-X₁₉-W and F/I-X₁₈-W-X₁₈-W). The black background represents a 100% consistent sequence. Red and blue represent 75% and 50% respectively. (C) Phylogenetic tree analysis of LmMYB1 (noted with a red star) and homologous in other plants.

Figure 2

Gene expression profile of L. multiflorum under abiotic stress. (A) Related genes of the ABA biosynthetic pathway and LmMYB1 under drought stress in the previous transcriptome data [11]. The ordinate represents the fold change (CK vs. DR) of genes under drought stress. There are three biological replicates per treatment, with the average shown in the figure. The color from blue to red indicates low to high gene expression. The expression profile of LmMYB1’s response to drought stress (B) and ABA treatment (C) in ‘Chuannong No. 1′. Three biological repetitions per time point, three technical repetitions. The standard deviation is represented by the error bar. LmHIS3 and LmeIF4A were used as the reference genes and qRT-PCR data were analyzed using the 2^−∆∆Ct method. The asterisk indicates the difference compared to 0 h (* p < 0.05, ** p < 0.01).

Figure 3

Comparison of abiotic stress tolerance of overexpressing LmMYB1 lines with wild type on 1/2 MS medium. (A) Phenotype of the WT and OE lines under 5 μM ABA, 200 mM mannitol and 100 mM NaCl compared to 1/2 MS. (B,C) Fresh weight and root length of the WT and OE lines under different treatments, n = 15, * p < 0.05, ** p < 0.01, **** p < 0.0001, ns, non-significant.

Figure 4

Overexpression of LmMYB1 can improve drought tolerance in A. thaliana. (A) Phenotype of the WT and transgenic lines (OE1, OE4, OE9) under drought stress. (B) Survival rate of the WT and transgenic lines after re-watering. (C–G) Shows measurements of tolerance-related physiological parameters. Data are presented as mean and SD values of four independent experiments. Asterisks indicate significant difference (* p < 0.05, ** p < 0.01, by independent sample t-test) between the WT and OE lines.

Figure 5

Analysis of stress resistance gene expression in WT and overexpressed LmMYB1 under drought stress. The value is the average of three biological repetitions and three technical repetitions. The standard deviation is represented by the error bar. AtACT2 was used as the reference gene and qRT-PCR data were analyzed using the 2^−∆∆Ct method. The asterisk indicates the difference compared to WT (* p < 0.05, ** p < 0.01).

Figure 6

GO enrichment analysis and core DEGs heat map of WT vs. OE gene. (A,B) The top ten terms enriched by up-regulated and down-regulated genes were shown, respectively. The vertical axis represents different biological processes, and the horizontal axis represents rich factors. The size of the dots represents the number of genes enriched. The color of the dots represents the -log₁₀(Qvalue) of the event. (C) The heat map of core genes. The core gene is derived from the biological process enriched in the previous step. The right ordinate is the number and name of different genes. (D) Validation of transcriptome data of key genes. In qRT-PCR, each square is formed by three technical repetitions and AtACT2 was used as the reference gene. The right ordinate shows the name of the gene and its approximate functional range. The color from blue to red indicates low to high gene expression.

Figure 7

Overexpression of LmMYB1 decreased stomatal density in A. thaliana. (A–C) Representation figure of stomatal density of the WT and transgenic lines (OE1, OE4). The red circle represents each stoma. Scale = 1 μm. (D) Statistics of stomatal density of the WT and transgenic lines (OE1, OE4). There were three biological replicates per line and 10 visual fields (10 × 40) were collected for each biological replicate. Asterisks indicate significant difference (** p < 0.01, *** p < 0.001, by independent sample t-test) between WT and OE lines.

Figure 8

Hypothetical model of the LmMYB1-regulated network in the drought response.