The C-Terminal Region of SLIM1 Transcription Factor Is Required for Sulfur Deficiency Response

Abstract

Sulfur LIMitation1 (SLIM1) transcription factor coordinates gene expression in plants in response to sulfur deficiency (-S). SLIM1 belongs to the family of plant-specific EIL transcription factors with EIN3 and EIL1, which regulate the ethylene-responsive gene expression. The EIL domains consist of DNA binding and dimerization domains highly conserved among EIL family members, while the N- and C-terminal regions are structurally variable and postulated to have regulatory roles in this protein family, such that the EIN3 C-terminal region is essential for its ethylene-responsive activation. In this study, we focused on the roles of the SLIM1 C-terminal region. We examined the transactivation activity of the full-length and the truncated SLIM1 in yeast and Arabidopsis. The full-length SLIM1 and the truncated form of SLIM1 with a deletion of C-terminal 106 amino acids (ΔC105) transactivated the reporter gene expression in yeast when they were fused to the GAL4 DNA binding domain, whereas the deletion of additional 15 amino acids to remove the C-terminal 120 amino acids (ΔC120) eliminated such an activity, identifying the necessity of that 15-amino-acid segment for transactivation. In the Arabidopsis slim1-2 mutant, the transcript levels of SULTR1;2 sulfate transporter and the GFP expression derived from the SULTR1;2 promoter-GFP (P_SULTR1;2-GFP) transgene construct were restored under -S by introducing the full-length SLIM1, but not with the C-terminal truncated forms ΔC105 and ΔC57. Furthermore, the transcript levels of -S-responsive genes were restored concomitantly with an increase in glutathione accumulation in the complementing lines with the full-length SLIM1 but not with ΔC57. The C-terminal 57 amino acids of SLIM1 were also shown to be necessary for transactivation of a -S-inducible gene, SHM7/MSA1, in a transient expression system using the SHM7/MSA1 promoter-GUS as a reporter. These findings suggest that the C-terminal region is essential for the SLIM1 activity.

Keywords: Arabidopsis thaliana; SLIM1 transcription factor; sulfate assimilation; sulfate transporter; sulfur deficiency.

Figure 1

Alignment of SLIM1 and EIN3 proteins in Arabidopsis thaliana. Alignment of full protein sequences was performed by the ClustalW program at the DNA Data Bank of Japan (DDBJ) (http://www.ddbj.nig.ac.jp/search/clustalw-j.html, accessed on 8 May 2017). Amino acid residues conserved between SLIM1 and EIN3 are shown with white characters and dark gray background. Predicted DNA binding domains (BD I to BD V) are shown with pale blue bars. Yellow and brown characters indicate the positions of slim1 mutations (slim1-1, slim1-2, slim1-3, and slim1-4) and ein3-3 mutation, respectively. Magenta characters show the last amino acid in each C-terminal truncated form of SLIM1. Green and orange bars describe existing knowledge about domain structures of SLIM1 and EIN3, respectively. Violet bars highlight the findings in this study.

Figure 2

Trans-activation of HIS3 gene expression in yeast. The left panel shows the schematic representations of SLIM1 proteins fused to the GAL4 DNA-binding domain (BD-GAL4). Empty: no insertion, SLIM1: full-length SLIM1, ΔC57, ΔC105, ΔC120, ΔC137, ΔC150, and ΔC165: C-terminal truncated forms of SLIM1 lacking the C-terminal 57, 105, 120, 137, 150, and 165 amino acids, respectively. BD-GAL4: DNA binding domain of yeast GAL4 protein. Four independent colonies obtained from each plasmid transformation of yeast strain AH109 were spotted on the minimal SD agar medium lacking Trp (−Trp) or Trp and His (−Trp/−His) with or without 10 mM 3-amino-1,2,4-triazole (3-AT). The right panel shows the yeast growth at 30 °C for 3 days after spotting.

Figure 3

Complementation of Arabidopsis slim1-2 by expressing the full-length and C-terminal truncated forms of SLIM1. The full-length SLIM1 or the C-terminal truncated variants were expressed in slim1-2 under CaMV 35S promoter (SLIM1/slim1-2, ΔC57/slim1-2, and ΔC105/slim1-2). Parental (P_SULTR1;2-GFP), slim1-2, and four independent lines of SLIM1/slim1-2, ΔC57/slim1-2, and ΔC105/slim1-2 were grown on S1500, S15, and S0 media for 10 days. (a) GFP fluorescence in plants is visualized using an image analyzer. Fluorescent images (upper panels) and bright-field images (lower panels) are shown. (b) Transcript levels of SULTR1;2 in roots. The average values are indicated with error bars denoting SEM (n = 3) for Parental and slim1-2 (left), and the single values are indicated for four independent transgenic lines generated for complementation with the full-length SLIM1 or the C-terminal truncated variants. Asterisks indicate significant differences between Parental and slim1-2 determined by Student’s t-test (left), and between SLIM1/slim1-2 and ΔC57/slim1-2 or ΔC105/slim1-2 by Dunnet’s test (right) under S1500 and S0 conditions (** p < 0.01, * 0.01 ≤ p < 0.05).

Figure 4

Transcript levels of −S-responsive genes in the complemented lines. Transcript levels of BGLU28, SDI1, SULTR2;1, and APR3 in the roots of four independent lines of SLIM1/slim1-2 and ΔC57/slim1-2 were determined by qRT-PCR with the same root-derived RNA used for the SULTR1;2 transcript expression analysis in Figure 3b. Asterisks indicate significant differences between SLIM1/slim1-2 and ΔC57/slim1-2 in S1500 and S0 conditions (Student’s t-test; ** p < 0.01, * 0.01 ≤ p < 0.05).

Figure 5

Sulfate, cysteine, and glutathione (GSH) levels in the complementation lines. Shoots of Parental (P_SULTR1;2-GFP), slim1-2, and four independent lines of SLIM1/slim1-2 and ΔC57/slim1-2 grown on S1500 and S0 media for 10 days were used for the metabolite analysis. The average values are indicated with error bars denoting SEM (n = 3) for Parental and slim1-2, and the single values are indicated for four independent transgenic lines generated for complementation with the full-length SLIM1 or ΔC57. Asterisks indicate significant differences between Parental and slim1-2 (left), and between SLIM1/slim1-2 and ΔC57/slim1-2 (right) under S1500 and S0 conditions (Student’s t-test; ** p < 0.01, * 0.01 ≤ p < 0.05).

Figure 6

Transactivation of SHM7/MSA1 promoter by full-length and C-terminal 57 aa truncated SLIM1. GUS activity was measured in transiently transformed Nicotiana benthamiana leaves incubated for 72 h after the infiltration with the reporter (SHM7/MSA1 promoter-GUS) or the combination of the reporter and effectors (SLIM1 or ΔC57). The uidA gene in the reporter constructs was driven by the SHM7/MSA1 promoter (569 bp upstream of ATG), and the effector expressions were driven by the CaMV 35S promoter. The mean values are indicated with error bars denoting SD (n = 3). Asterisks indicate significant differences between the two combinations of reporter and effector transfected in each leaf (Student’s t-test; ** p < 0.01).