Volume 10, Issue 5, Pages 735-748 (May 2017) Arabidopsis Transcription Factors SPL1 and SPL12 Confer Plant Thermotolerance at Reproductive Stage  Lu-Men Chao, Yao-Qian Liu, Dian-Yang Chen, Xue-Yi Xue, Ying-Bo Mao, Xiao-Ya Chen  Molecular Plant  Volume 10, Issue 5, Pages 735-748 (May 2017) DOI: 10.1016/j.molp.2017.03.010 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Functional Domain Analysis and Expressions of SPL1 and SPL12 in Arabidopsis. (A) Schematic of functional domains of SPL1 and SPL12 predicted by SMART. The SBP box (SBP), ankyrin domain (ANK), and transmembrane domain (T) are indicated. (B) Subcellular localization of the SPL1. EYFP was in-frame fused to the N terminus of SPL1 with (EYFP-SPL1) or without (EYFP-SPL1ΔT) the transmembrane domain, and transiently expressed in N. benthamiana leaf cells via Agrobacterium infiltration. The nucleus stained by DAPI is displayed in the inset. Scale bars, 20 μm. (C) Relative expressions of SPL1 and SPL12 in different organs. Sd, seedling; Rl, rosette leaves; St, stem; lnf, inflorescence; Sp, sepal; Pt, petal; Re, reproductive organs (stamen and pistil). Gene expressions were analyzed by qRT-PCR, S18 was used as an internal reference, the relative expression level of SPL1 in Rl was set to 1, and values are means ± SEM (n = 3). (D) GUS staining of the pSPL1::GUS and pSPL12::GUS in Arabidopsis inflorescence. Scale bars, 1 mm. Molecular Plant 2017 10, 735-748DOI: (10.1016/j.molp.2017.03.010) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Reduced Thermotolerance of spl1-1 spl12-1 Inflorescences. (A) Schematic of A. thaliana SPL1 (AT2G47070) and SPL12 (AT3G60030) genes. The SPL1 and SPL12 coding regions are composed of 2646 bp and 2784 bp for proteins of 881 and 927 amino acids, respectively. Line indicates intron, box indicates exon, and SBP box is black. Black arrow indicates the T-DNA insertion site in spl1-1 and spl12-1; orange or blue arrows indicate RT–PCR primer sites for expression analysis. (B) View of inflorescences after heat treatment, categorized as plump (normal), wilted (partly burned or drooping), and scorched (burned or dried out, unable to recover). Scale bars, 2 mm. (C) Percentages of each category in the wild-type (Col-0), spl1-1, spl12-1, and spl1-1 spl12-1 lines. Plants were treated at 42°C for 5 h, followed by phenotype examination immediately. Plants of all genotypes had nearly 100% plump under 22°C. ***P < 0.001 compared with the wild-type by Fisher's exact test (n = 80–100 inflorescence from 24 plants). (D) Change of SOD activity in inflorescences after the heat treatment. Values in box plots are means ± SEM (n = 3, 20 inflorescences each), ***P < 0.001 compared with the wild-type under 42°C by multiple comparisons (Bonferroni post-test) followed by one-way ANOVA. The interaction between two factors (line and temperature) is also significant (**P < 0.01). (E) H2O2 accumulation detected by DAB reaction. Scale bars, 1 cm. (F) Trypan blue staining of cell death. Scale bars, 1 cm. (G) Seed yields. Plants were maintained at 22°C and treated with heat (42°C, 5 h) at 35 days post germination. Seed production per plant was recorded; values are shown in box plots (n = 36, left). Right: ***P < 0.001 compared as that in (D) (comparisons between the wild-type and spl1-1 spl12-1 under 22°C/42°C). Molecular Plant 2017 10, 735-748DOI: (10.1016/j.molp.2017.03.010) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Overexpression of SPL1 or SPL12 Enhances Plant Thermotolerance at the Reproductive Stage. 35::Myc-SPL1 was expressed in wild-type Col-0 (ox-S1c) or spl1-1 spl12-1 double-mutant (ox-S1d) background, and 35::SPL12 was expressed in Col-0 (ox-S12c) background. (A) View of the 5-week-old plants grown at 22°C (top) or subjected to heat (42°C, 5 h) treatment (bottom). Scale bars, 5 cm. (B) Enlarged view of the inflorescence shown in (A) as numbered in red. Scale bars, 2 cm. (C) Percentages of three categories as in Figure 2B in the wild-type (Col-0) and overexpressors. The heat treatment caused less damage to inflorescences of the overexpressors. *P < 0.05, ***P < 0.001 compared with the wild-type by Fisher's exact test (n = 80–100 inflorescences from 24 plants for each line of each test). (D) SPL1 and SPL12 overexpression increased the seed yields. Plants were treated as in Figure 2G, seed yields were recorded and are shown in box plots (n = 18); *P < 0.05, **P < 0.01, ***P < 0.001 compared with the wild-type under relevant temperature by multiple comparisons (Bonferroni post-test) followed by one-way ANOVA. The interaction between two factors (line and temperature) is also significant (Supplemental Figure 5). Molecular Plant 2017 10, 735-748DOI: (10.1016/j.molp.2017.03.010) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Overexpression of SPL1 or SPL12 Improves the Performance of Tobacco (N. benthamiana) Flowers under Heat Stress. (A) View of transgenic N. benthamiana plants expressing 35::Myc-SPL1 (S1) and 35::SPL12 (S2) grown at 22°C (top) or subjected to heat treatment at 45°C (bottom). The 4-week-old tobacco plants were subjected to a 6-h heat treatment under 45°C followed by recovery for 2 weeks. Scale bars, 5 cm. (B) View of flowers of the indicated plants after recovery. Scale bars, 5 cm. (C) Survival rates of tobacco flowers. The percentage of viable flowers among a total of nine flowers each plant was calculated after the recovery; 10 plants in each line were analyzed. **P < 0.01 compared with the wild-type by multiple comparisons (Bonferroni post-test) followed by one-way ANOVA. Molecular Plant 2017 10, 735-748DOI: (10.1016/j.molp.2017.03.010) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Transcriptomic Profiling of Arabidopsis Inflorescences Treated with Heat. (A) Heat-responsive genes in inflorescences of indicated plants. Inflorescences of the 5-week-old wild-type (wt) and spl1-1 spl12-1 (dm) plants grown at 22°C (control) or treated with heat (42°C, 1 h) were analyzed with three biological replicates. Red dots indicate genes significantly up- or downregulated upon heat treatment, and numbers of the genes are denoted in the upper right (upregulated) and left (downregulated) corner of each panel. (B) Significantly enriched GO terms of the heat-responsive upregulated genes in the wild-type inflorescences using AgriGO. GO terms were selected with false discovery rate <0.05 by hypergeometric test. Red line indicates P value of 0.01. (C) Heatmap of differentially expressed genes of indicated samples. Pink, upregulation; blue, downregulation; white, intermediate. Molecular Plant 2017 10, 735-748DOI: (10.1016/j.molp.2017.03.010) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Heat-Responsive Differently Expressed Genes in spl1-1 spl12-1 Inflorescence. (A) Heat-responsive differently expressed genes (HRD) are classified into three classes: (1) HRD-Up, including wt+/dmN (upregulated in wild-type but not in spl1-1 spl12-1) and wt+>dm+ (upregulated more in wild-type than in spl1-1 spl12-1); (2) HRD-down, including wt−/dmN (downregulated in wild-type but not in spl1-1 spl12-1) and wt−<dm− (downregulated more in wild-type than in spl1-1 spl12-1); (3) HRD-Neutral, including wtN/dm+ (unchanged in wild-type but upregulated in spl1-1 spl12-1) and wtN/dm− (unchanged in wild-type but downregulated in spl1-1 spl12-1). (B) GO-term enrichment in HRD-Up using AgriGO (blue). Gray bars indicate the percentage of enrichment across the whole genome. Asterisks indicate −log(P-value) by hypergeometric test. (C) Enrichments of cis elements in promoters of HRD-Up genes (Green). Gray bars indicate the percentage of enrichment across the whole genome. Asterisks indicate −log(P-value) by hypergeometric test. (D) Transcriptional activation of target genes by SPL1. Promoters were fused to the LUC reporter, and the activities were determined by transient dual-LUC assay in tobacco leaf. The relative LUC activities were normalized to REN; pro, promoter region; data are presented as mean ± SEM, n = 3; *P < 0.05, ***P < 0.001, Student's t-test. Molecular Plant 2017 10, 735-748DOI: (10.1016/j.molp.2017.03.010) Copyright © 2017 The Authors Terms and Conditions

Figure 7 SPL1 and SPL12 Are Involved in Heat Induction of a Subset of ABA-Responsive Genes. (A) Assay of thermotolerance of ABA mutants. Percentages of three categories as in Figure 2B are shown. *P < 0.05, ***P < 0.001. (B) Venn diagram depicting the overlap of upregulated genes in response to heat or ABA. (C) Relative expressions of 10 HRD-up ABA-responsive genes in the wild-type, spl1-1 spl12-1, and pyl sextuple mutant inflorescences upon heat treatment (42°C for 0, 1, and 4 h). (D) Relative expressions of HRD-up ABA-responsive genes in wild-type inflorescences after 4-h ABA (100 μM) application. Gene expressions were analyzed by qRT-PCR, and S18 was used as reference. Values are means ± SEM (n = 3). Molecular Plant 2017 10, 735-748DOI: (10.1016/j.molp.2017.03.010) Copyright © 2017 The Authors Terms and Conditions

Figure 8 SPL1 and SPL12 Require PYL-Mediated ABA Signaling to Confer Inflorescence Thermotolerance. (A) Effects of ABA pretreatment. Inflorescences were pretreated with 100 μM ABA and 4 h later the plants were treated with heat at 42°C for 5 h (ABA + 42°C). Percentages of three categories as in Figure 2B are shown. Pretreatments with (H2O + 42°C) or without (CK + 42°C) H2O were used as controls. ***P < 0.001 compared with the wild-type by Fisher's exact test (n = 80–100 inflorescences from 24 plants for each treatment and each line). (B) SPL1 and SPL12 overexpression improved inflorescence thermotolerance in aba2-1 but not in pyl sextuple mutant. **P < 0.01 compared with aba2-1 by Fisher's exact test (n = 80–100 inflorescences from 24 plants for each line). It was 100% (or nearly so) plump under 22°C for all genotypes tested in (A) and (B). Molecular Plant 2017 10, 735-748DOI: (10.1016/j.molp.2017.03.010) Copyright © 2017 The Authors Terms and Conditions