Volume 9, Issue 9, Pages 1337-1340 (September 2016) Soybean miR172a Improves Salt Tolerance and Can Function as a Long-Distance Signal  Wen-Jia Pan, Jian-Jun Tao, Tong Cheng, Xiao-Hua Bian, Wei Wei, Wan-Ke Zhang, Biao Ma, Shou-Yi Chen, Jin-Song Zhang  Molecular Plant  Volume 9, Issue 9, Pages 1337-1340 (September 2016) DOI: 10.1016/j.molp.2016.05.010 Copyright © 2016 The Author Terms and Conditions

Figure 1 Soybean miR172a Improves Salt Tolerance as a Long-Distance Signal by Degrading the SSAC1, an AP2-type Transcription Suppressor Gene that Restrains the Thiamin Biosynthesis-Related Gene THI1. (A) Performance of soybean plants harboring 172a-OHR (pre-miR172a-overexpressing hairy roots) or CHR (control hairy roots) under normal and salt stress conditions. Control hairy roots are generated after Agrobaterium rhizogenis (strain K599) infection. More than 30 lines were analyzed, and typical lines are shown. Scale bar, 2 cm. (B) miR172a expression in CHR and 172a-OHR plants by qRT–PCR. (C) Root elongation and shoot elongation of CHR and 172a-OHR plants. (D) Expression of the miR172a target gene SSAC1 by qRT–PCR. (E) Performance of soybean plants harboring SSAC1-OHR (overexpressing hairy root) and SSAC1-RHR (RNAi hairy root). Scale bar, 2 cm. (F) Transcript level of SSAC1 in different transgenic hairy roots. (G) Root elongation and shoot elongation of SSAC1-OHR and SSAC1-RHR plants. (H) Performance of plants harboring different overexpressing transgenic hairy roots under salt stress. mSSAC1 is SSAC1 with a mutation at the miR172a cleavage site but without amino acid change. Scale bar, 2 cm. (I) Transcript level of SSAC1 in plants with different overexpressing transgenic hairy roots. (J) Root elongation and shoot elongation of plants with different overexpressing transgenic hairy roots. (K) Dual-luciferase reporter assay of the SSAC1 protein activity in transcriptional regulation using an Arabidopsis protoplast system (Hao et al., 2011). (L) SSAC1 suppressed the transcriptional activity of THI1 promoter when transiently expressed in tobacco leaves. Quantitative analysis of the luminescence intensity is shown on the right for comparison. (M) Gel-shift assay showed that SSAC1 can bind to the THI1 promoter. P, labeled probe; mP, labeled probe with mutated motif. (N) Performance of plants harboring THI1-OHR and THI1-RHR. (O) Transcript level of THI1 in different transgenic hairy roots. (P) Root elongation and shoot elongation of THI1-OHR and THI1-RHR plants. (Q) Phenotype of different grafted plants. For each panel, the yellow box indicates the grafting site. Control, WT scions were grafted on CHR stocks harboring control hairy roots. Graft, WT scions were grafted on the 172a-OHR stocks harboring pre-miR172a-overexpressing hairy roots. The right panels are the enlargement of the grafting site. (R) qRT–PCR analysis of miR172a and SSAC1 expression pattern in different grafted plants. More than six graft plants were detected and shown as means ± SD. (S) Model of miR172a function in plant response to salt stress. Under salt stress, soybean miR172a was induced and can function as a transportable signal from root to shoot. In both roots and shoots, miR172a cleaves its target gene SSAC1, which encodes a transcriptional repressor to inhibit the THI1 expression. Finally, miR172a-promoted THI1 expression enhances thiamine biosynthesis for plant tolerance to salt stress. For qRT–PCR, values are normalized to tubulin beta1 and shown as means ± SD (n = 4). For measurements of root elongation and shoot elongation, values are means ± SD (n ≥ 30). For detection of LUC activity, values are means ± SD (n = 3). Salt stress treatment in all experiments was 80 mM NaCl. All experiments were performed at least three times with similar results and one typical result is shown. Asterisks indicate significant differences compared with the corresponding controls (t-test; *P < 0.05; **P < 0.01). Molecular Plant 2016 9, 1337-1340DOI: (10.1016/j.molp.2016.05.010) Copyright © 2016 The Author Terms and Conditions