Volume 8, Issue 10, Pages 1536-1549 (October 2015) Disruption of the Arabidopsis Defense Regulator Genes SAG101, EDS1, and PAD4 Confers Enhanced Freezing Tolerance  Qin-Fang Chen, Le Xu, Wei-Juan Tan, Liang Chen, Hua Qi, Li-Juan Xie, Mo-Xian Chen, Bin-Yi Liu, Lu-Jun Yu, Nan Yao, Jian-Hua Zhang, Wensheng Shu, Shi Xiao  Molecular Plant  Volume 8, Issue 10, Pages 1536-1549 (October 2015) DOI: 10.1016/j.molp.2015.06.009 Copyright © 2015 The Author Terms and Conditions

Figure 1 The sag101, eds1, and pad4 Mutants Show Enhanced Tolerance to Freezing. (A) Images of non-acclimated (NA) wild type (WT), sag101-3, eds1-22, and pad4-1 mutants before (CK) and after 1 h treatment at various freezing temperatures (−6, −8, and −10°C), followed by 7-day recovery at normal growth conditions. (B and C) Survival rate (B) and dry weight (C) of NA WT, sag101-3, eds1-22, and pad4-1 plants after freezing treatment (−6, −8, and −10°C) followed by 7-day recovery. (D) Electrolyte leakage of NA WT, sag101-3, eds1-22, and pad4-1 plants after freezing treatment (−3, −4, −5, −6, −7, and −8°C). (E) Images of cold-acclimated (CA) WT, sag101-3, eds1-22, and pad4-1 plants before (CK) and after 1 h treatment at various freezing temperatures (−8, −10, and −12°C), followed by 7-day recovery at normal growth conditions. (F and G), Survival rate (F) and dry weight (G) of CA WT, sag101-3, eds1-22, and pad4-1 plants after freezing treatment (−8, −10, and −12°C) followed by 7-day recovery. (H) Electrolyte leakage of CA WT, sag101-3, eds1-22, and pad4-1 plants after freezing treatment (−7, −8, −9, −10, −11, and −12°C). The experiments have been repeated three times with biological replicates and >15 plants were used for each experiment. Data are average values ± SD (n = 3) calculated from three independent experiments. Asterisks indicate significant differences from wild type (*P < 0.05; **P < 0.01 by Student’s t-test). Molecular Plant 2015 8, 1536-1549DOI: (10.1016/j.molp.2015.06.009) Copyright © 2015 The Author Terms and Conditions

Figure 2 Cold-Responsive Expression of CBFs and Accumulation of Osmolytes in Wild Type, sag101-3, eds1-22, and pad4-1. (A) Expression of CBF genes and their regulons in wild type (WT), sag101-3, eds1-22, and pad4-1 plants under cold stress (4°C). Total RNA samples were extracted from 4-week-old WT, sag101-3, eds1-22, and pad4-1 plants treated at 4°C for 0, 1, 3, 6, 12, and 24 h. The transcripts of CBF1, CBF2, CBF3, COR47, RD29A, and KIN1 were analyzed by qRT–PCR. The 2−▵▵Ct method (Livak and Schmittgen, 2001) was used to calculated the cycle threshold (Ct) value of each sample. The expression level changes of CBF1, CBF2, CBF3, COR47, RD29A, and KIN1 were normalized with ACTIN2. The relative expression of these genes is presented as 2−▵▵Ct. The experiments were repeated (biological replicates) three times, with similar results, and the representative data from one replicate are shown. Data are means ± SD (n = 3) of three technical replicates. Asterisks indicate significant differences from wild type (**P < 0.01 by Student’s t-test). (B) Contents of proline and soluble sugars in WT, sag101-3, eds1-22, and pad4-1 plants under NA and CA conditions. WT, sag101-3, eds1-22, and pad4-1 plants were untreated (NA) or cold-treated for 3 days and rosettes were collected for metabolite extraction. Ribitol was added as an internal quantitative standard for GC-MS analysis. The experiments were repeated (biological replicates) three times, with similar results, and the representative data from one replicate are shown. Data are means ± SD calculated from three technical replicates. Asterisks indicate significant differences from wild type (*P < 0.05; **P < 0.01 by Student’s t-test). Molecular Plant 2015 8, 1536-1549DOI: (10.1016/j.molp.2015.06.009) Copyright © 2015 The Author Terms and Conditions

Figure 3 Cell Death and ROS Levels in Wild-Type (WT), sag101-3, eds1-22, and pad4-1 Rosettes upon Freezing Treatment. (A) Trypan blue staining showing cell death in the rosettes of WT, sag101-3, eds1-22, and pad4-1 plants. Bars represent 1 mm. (B) DAB staining showing ROS accumulation in the rosettes of WT, sag101-3, eds1-22, and pad4-1 plants. Bars represent 1 mm. Four-week-old WT, sag101-3, eds1-22, and pad4-1 plants were untreated (NA) or cold-acclimated for 3 days (CA) then transferred to −6°C (for NA plants) or −8°C (for CA plants) for 1 h and subsequently recovered for 12 h at 4°C. Rosettes were collected for trypan blue and DAB staining. Molecular Plant 2015 8, 1536-1549DOI: (10.1016/j.molp.2015.06.009) Copyright © 2015 The Author Terms and Conditions

Figure 4 Levels of Free SA and SA Glucosides in the Rosettes of Wild-Type (WT), sag101-3, eds1-22, and pad4-1 Plants. Free SA and SA glucosides were extracted from rosettes of 4-week-old WT, sag101-3, eds1-22, pad4-1, and siz1 plants harvested after NA, CA, freezing treatment (−8°C) for 1 h (Freezing) and recovery for 12 h (Recovery), and the extracts were analyzed by LC-MS/MS. The experiments were repeated (biological replicates) three times, with similar results, and the representative data from one replicate are shown. Data are means ± SD calculated from three technical replicates. Asterisks with “a” indicate significant differences from that of NA wild type and with “b” indicate significant differences from wild type (*P < 0.05; **P < 0.01 by Student’s t-test). Molecular Plant 2015 8, 1536-1549DOI: (10.1016/j.molp.2015.06.009) Copyright © 2015 The Author Terms and Conditions

Figure 5 Lipid Profiles of 4-Week-Old Wild-Type (WT), sag101-3, eds1-22, and pad4-1 Rosettes Before (22°C) and After CA Followed by Freezing Treatment (−8°C). (A) Freezing-induced changes in the compositions of phospholipids (PC, PE, PI, PS, and PA) of WT, sag101-3, eds1-22, and pad4-1 rosettes before (22°C; left column) and after CA followed by freezing treatment (−8°C; right column). (B and C) Total amounts (signal/mg dry weight) of diacylglycerol (DAG; left column) and triacylglycerol (TAG; right column) in WT, sag101-3, eds1-22, and pad4-1 rosettes. The DAG/TAG ratio of each treatment is shown in (C). Data are means ± SD calculated from four technical replicates. Asterisks indicate significant differences from wild type (*P < 0.05; **P < 0.01 by Student’s t-test). Molecular Plant 2015 8, 1536-1549DOI: (10.1016/j.molp.2015.06.009) Copyright © 2015 The Author Terms and Conditions

Figure 6 Effect of Supplementation with SA, DAG, and PA on Freezing Sensitivities of sag101-3, eds1-22, and pad4-1 Mutants. Four-week-old wild-type (WT), sag101-3, eds1-22, and pad4-1 plants were sprayed with dH2O, 200 μM SA (Sigma-Aldrich), DAG (1,2-dioctanoyl-sn-glycerol from Avanti Polar Lipids), and PA (mixture from Sigma-Aldrich) liposomes and incubated under normal growth conditions for 24 h. The plants were subsequently treated with freezing temperature (−8°C) for 1 h followed by 7-day recovery. Molecular Plant 2015 8, 1536-1549DOI: (10.1016/j.molp.2015.06.009) Copyright © 2015 The Author Terms and Conditions

Figure 7 Effect of SA on the Expression of Genes Involved in DAG Metabolism in Wild-Type Plants under Cold (4°C) and Freezing (−8°C) Stresses. Total RNA samples were extracted from 4-week-old wild-type plants treated with dH2O (−SA) or 200 μM SA (+SA) at 4°C for 0, 1, 3, 6, 12, and 72 h as well as freezing (−8°C) for 1 h (F1) following recovery at 4°C for 6 h (R6). The expression level changes of DGATs (DGAT1, DGAT2, and DGAT3) and DGKs (DGK2, DGK3, and DGK5) were normalized with ACTIN2. The relative expression levels of these genes were presented as 2−▵▵Ct. The experiments were repeated (biological replicates) three times, with similar results, and the representative data from one replicate are shown. Data are means ± SD (n = 3) of three technical replicates. Asterisks indicate significant differences of SA treatment from that of dH2O treatment (*P < 0.05; **P < 0.01 by Student’s t-test). “a” and “b” indicate decreased and increased, respectively, gene expression levels in the SA-treated samples in comparison with the corresponding dH2O-treated controls. Molecular Plant 2015 8, 1536-1549DOI: (10.1016/j.molp.2015.06.009) Copyright © 2015 The Author Terms and Conditions