A WD40-Repeat Protein From the Recretohalophyte Limonium bicolor Enhances Trichome Formation and Salt Tolerance in Arabidopsis

Abstract

The Arabidopsis thaliana WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1) controls epidermis development, playing opposite roles in trichome differentiation and root hair formation. We isolated and characterized LbTTG1 (encoding a WD40-repeat protein with high sequence similarity to TTG1) from the recretohalophyte Limonium bicolor, which actively excretes absorbed salt via a salt gland. The complete open reading frame of LbTTG1 was 1,095 bp, encoding a protein of 364 amino acids, and showed highest expression during the salt gland initiation stage. We heterologously expressed LbTTG1 in wild type and ttg1-13 Arabidopsis plants to verify the protein's function, and the copies of LbTTG1 were identified in transgenic strains using southern blotting. Trichomes were extremely induced on the first true leaves of plants heterologously expressing LbTTG1, whereas no trichomes were produced by ttg1-13 plants. Conversely, plants heterologously expressing LbTTG1 produced fewer root hairs than ttg1-13 plants. In plants heterologously expressing LbTTG1 compared to controls, epidermis differentiation genes (GLABRA1 and GLABRA3) were up-regulated while genes encoding negative regulators of trichome development (TRIPTYCHON and CAPRICE) were down-regulated. Under increased NaCl concentrations, both of the transgenic lines showed enhanced germination and root length, and accumulated less malondialdehyde (MDA) and Na⁺ and produced more proline, soluble sugar, and higher glutathione S-transferase activity, compared with the ttg1-13 mutant. These results indicate that LbTTG1 participates in epidermis development in Arabidopsis, similarly to other WD40-repeat proteins, and specifically increases salt tolerance of transgenic Arabidopsis by reducing ion accumulation and increasing osmolyte levels.

Keywords: Arabidopsis; Limonium bicolor; WD40-repeat protein; heterologous expression; root hair; salt stress; trichome.

Figure 1

LbTTG1 encoded WD40 repeat protein. (A) Nucleotide and amino acid sequences of LbTTG1 analyzed with DNAman. The yellow shadows indicated four WD-repeat domains. (B) The sequence alignment of TTG1 between Limonium bicolor and Arabidopsis. The identity is 73.9%. (C) Four conserved WD-repeat domains of LbTTG1, located in 86∼132, 139∼184, 187∼225, and 276∼316 amino acids; pink mean the low complexity domain, drawn with SMART. (D) Phylogenetic relationships of different plants based on amino acid residues of LbTTG1, reconstructed by the neighbor-joining method using MEGA and ClustalX software. Bar, 0.05 substitutions per amino acid position.

Figure 2

Subcellular localization by onion epidermis transformation and expression level of LbTTG1 at different development stages and conditions. (A) Subcellular localization of LbTTG1 after transformation of onion epidermis. GFP-LbTTG1 fusion protein was expressed in both the nucleus and cytoplasm. 35S::GFP is the empty control vector, and 35S::LbTTG1-GFP is the experiment group. Bars = 50 µm. (B) Expression levels of LbTTG1 at different developmental stages and conditions. Stages A-E were the continues leaf development stage. Stage A, undifferentiation, 4–5 days after sowing; stage B, salt gland differentiation, 6–7 days; stage C, stomata differentiation, 8–10 days; stage D, epidermis differentiation, 11–16 days; stage E, mature, ≥17 days after sowing. NaCl means the mature leaves isolated from the seedlings at stage E under 200 mM NaCl for 24 h. Roots were taken from the seedlings of stage E. Flower was the mixture of flower buds and complete flowers.

Figure 3

Identification of Arabidopsis lines heterologously expressing LbTTG1. (A) Genomic DNA PCR of Col 35S::LbTTG1 lines; lanes 1–6, different transgenic lines; lane 7, blank control with ddH₂O as template; lane 8, negative control with wild type DNA as template. (B) Genomic DNA PCR of ttg 35S::LbTTG1 lines (lanes 1–6). (C) Expression levels of LbTTG1 examined using quantitative PCR in Col 35S::LbTTG1 and ttg 35S::LbTTG1. L numbers represent lines of Col 35S::LbTTG1, e.g. L2 and L3; CL numbers represent complementation lines of ttg 35S::LbTTG1. Data are means of three replicates ± SD; different letters indicate significant differences at P = 0.05 according to Duncan’s multiple range test.

Figure 4

Southern hybridization of LbTTG1 in Limonium bicolor and transgenic lines of Arabidopsis. Lane M, DIG-labeled DNA molecular-weight marker; lane PC, positive control; lane Lb, genomic from L. bicolor; lane L3, L16, and L2, Arabidopsis Col 35S::LbTTG1; lane CL6, CL9, and CL3, Arabidopsis ttg 35S::LbTTG1. The red arrow indicated the possible gene copy.

Figure 5

Trichome development was enhanced after transformation of LbTTG1. (A) Trichomes on the first two rosette leaves of Col-0 wild type (WT), ttg1-13, Col 35S::LbTTG1 (L3, L16, and L2), and ttg 35S::LbTTG1 (CL6, CL9, and CL3). Photographs show 2-week-old soil-grown seedlings. (B) Trichome density on the first two rosette leaves of WT, ttg1-13, Col 35S::LbTTG1, and ttg 35S::LbTTG1. Data are mean ± SD of 10 plants; different letters indicate significant differences at P = 0.05 according to Duncan’s multiple range test.

Figure 6

Root hair development of Col 35S::LbTTG1 and ttg 35S::LbTTG1. (A) Phenotypes of root hairs of WT, ttg1-13, Col 35S::LbTTG1 (L3, L16, and L2), and ttg 35S::LbTTG1 (CL6, CL9, and CL3) after culture for 5 days on MS medium. (B) The same position of each root (region from 2 cm to 3 cm from root tip) was chosen for calculating root hair number and length from 10 or 20 plants from each line. Data for root hair number are mean ± SD of 10 plants. Data for root hair length are mean ± SD of 20 plants; different letters indicate significant differences at P = 0.05 according to Duncan’s multiple range test.

Figure 7

Germination of Col 35S::LbTTG1 and ttg 35S::LbTTG1 seeds under different NaCl concentrations. (A) Uniform, 1-day-old seedlings were transplanted to medium containing different NaCl concentrations (0, 50, 100, and 150 mM). Phenotypes of WT, ttg1-13, Col 35S::LbTTG1 (L3, L16, and L2), and ttg 35S::LbTTG1 (CL6, CL9, and CL3) in 5-day-old seedlings. (B) Germination percentage calculated 24 h after sowing on medium containing different NaCl concentrations (0, 50, 100, and 150 mM). Fifty seeds of each line were sowed in each treatment, and three replicates were performed. Data for germination percentage are mean ± SD. Root length of five-day-old seedlings was calculated using ImageJ software. Data for root length are mean ± SD of 20 plants per line; different letters indicate significant differences at P = 0.05 according to Duncan’s multiple range test.

Figure 8

The growth and biomass of different strains under different NaCl treatments. (A) Two-week-old seedlings of WT, ttg1-13, Col 35S::LbTTG1 (L3, L16, and L2), and ttg 35S::LbTTG1 (CL6, CL9, and CL3) under different NaCl treatments (0, 50, 100, and 150 mM). (B) Fresh weight and dry weight of intact seedlings under different NaCl treatments. Data are means ± SD of five replicates; different letters indicate significant differences at P = 0.05 according to Duncan’s multiple range test.

Figure 9

Ion contents, proline, MDA, GST activity, and soluble sugar contents of seedlings under different NaCl treatments. Seedlings under 0 and 100 mM NaCl treatments were pooled for measurement of Na⁺ and K⁺ contents, proline, MDA, GST activity, and soluble sugar contents. Data are means ± SD of five replicates; different letters indicate significant differences at P = 0.05 according to Duncan’s multiple range test.

Figure 10

Expression levels of trichome formation and stress-related marker genes. (A) Relative expression levels of AtTTG1, AtGL3, AtEGL3, AtGL1, AtCPC, and AtTRY in 5-day-old seedlings. (B) Relative expression levels of AtSOS1, AtSOS2, AtSOS3, AtAREB1, AtP5CS1, AtP5CS2, and AtGSTU5. Two-week-old seedlings of all lines germinated on MS medium were transferred to 1/2 MS liquid medium containing 100 mM NaCl for 0 and 3 h. Expression of each gene was measured in three replicate biological experiments; different letters indicate significant differences at P = 0.05 according to Duncan’s multiple range test.