1. Volume 9, Issue 10, Pages 1366-1378 (October 2016)
Control of Leaf Senescence by an MeOH-Jasmonates Cascade that Is Epigenetically Regulated by OsSRT1 in Rice 
ChuanYing Fang, Hua Zhang, Jian Wan, YangYang Wu, Kang Li, Cheng Jin, Wei Chen, ShouChuang Wang, WenSheng Wang, HaiWei Zhang, Pan Zhang, Fei Zhang, LiangHuan Qu, Xianqing Liu, Dao-Xiu Zhou, Jie Luo 
Molecular Plant 
Volume 9, Issue 10, Pages (October 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1 Natural Variation in Jasmonates Content.
(A) Heatmap of the natural variation of jasmonates in 221 rice varieties. The content value of each jasmonate is normalized and hierarchical clustering is performed. Each rice variety is visualized in a single column, and each jasmonate is represented by a single row. Yellow indicates high abundance jasmonates, whereas blue represents low relative abundance jasmonates.
(B) Boxplot for the content of three jasmonates in japonica (gray) and indica (white). The horizontal line represents the median, and the vertical lines mark the range from the 5th to the 95th percentile of the total data. FW, fresh weight. Two biological repeats were performed and asterisks indicate the levels of statistical significance as determined by Student's t test: **P < 0.01.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

3. Figure 2
Assignment of Possible Causative Sites for the Natural Variation in Jasmonates Content.
(A) Manhattan plots displaying the GWAS results for the content of three jasmonates. The horizontal dashed line indicates the thresholds set to P = 1.44E−6 by the LMM.
(B and C) Associations between polymorphic sites within the OsPME1 (B) and OsTSD2 (C) loci (MAF ≥0.05) and JA-Ile content. Each dot represents a polymorphic site.
(D and E) Representation of pairwise r2 values (a measure of LD) among all polymorphic sites in OsPME1 (D) and OsTSD2 (E); the darkness of the color of each box corresponds to the r2 value according to the legend. For the gene model, a filled black box represents the coding sequence.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

4. Figure 3
Natural Variation and Geographical Distribution of OsPME1 in China.
(A) Natural variation of OsPME1 in the 221 rice varieties. Nine haplotypes were detected, and the number of accessions in each haplotype is shown. The positions of SNPs are shown in the first row.
(B) Boxplot (the middle line indicates the median, the box indicates the range of the 5th to 95th percentiles of the total data, the whiskers indicate the interquartile range, and the outer dots are outliers) of JA-Ile content for the two classes. FW, fresh weight. The letter n indicates the number of accessions used for the analysis of the two haplotypes. Two biological repeats were performed.
(C) Boxplot (the middle line indicates the median, the box indicates the range of the 10th to 90th percentiles of the total data, the whiskers indicate the interquartile range, and the outer dots are outliers) of OsPME1 expression. RNA samples were collected from hydroponically grown 1-month-old plants in the greenhouse. The relative expression was analyzed by qRT–PCR with UBIQUITIN as an endogenous control. The letter n indicates the number of accessions used for the analysis of the two haplotypes. Three biological repeats were performed.
(D) Geographical distribution of the 221 accessions in China based on information from RiceVarMap ( The size of the pie chart is proportional to the sample size. The number of accessions in each area is represented as n.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

5. Figure 4
Transgenic Plants of OsPME1 and OsTSD2 Displayed Altered Jasmonates Accumulation and Gene Expression.
(A) Content of JA-Ile and OPDA in the wild-type and tsd2-1 plants.
(B) Content of JA-Ile in the wild-type and OsPME1.RNAi plants. Leaf samples were harvested from leaves at the tillering stage.
(C and D) Relative expression of JA biosynthetic and regulating genes in the tsd2-1 mutant (C) and OsPME1.RNAi plants (D) compared with that of the wild-type plants.
Values are given as the mean ± SD (n = 3). Mean values followed by * or ** were significant at the 0.05 or 0.01 probability level, respectively, compared with the wild-type. FW, fresh weight; WT, wild-type.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

6. Figure 5
Transgenic Plants of OsTSD2 and OsPME1 Displayed Altered Leaf Senescence.
(A) Delayed age-dependent leaf senescence was observed in the 120-day-old tsd2-1 mutant grown in the field (scale bar, 20 cm).
(B) Chlorophyll content of flag leaves from the wild-type plants and tsd2-1 mutants. The whole flag leaves from the first tiller of six plants were harvested and combined as one biological replicate of each sample.
(C) Expression of NOL and Osl43 in the wild-type plants and tsd2-1 mutants. The apical 3 cm of flag leaves from three plants were harvested and combined as one biological replicate of each RNA sample.
(D) Phenotype and chlorophyll content of OsPME1 transgenic and wild-type plants in the dark-induced leaf senescence assay. The apical 10 cm of the second upper leaves from 6-week-old plants were used for analysis.
In (B–D), data are shown as means ± SD. Three biological repeats were performed and asterisks indicate the levels of statistical significance as determined by Student's t test: **P < FW, fresh weight; WT, wild-type.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

7. Figure 6
MeOH Works Upstream of Jasmonates in the Control of Leaf Senescence.
(A) Content of MeOH in the flag leaves of 2-month-old wild-type and tsd2-1 plants.
(B) Phenotype of the wild-type and tsd2 mutants in the dark-induced leaf senescence assay under treatment of 1% MeOH (MeOH) or control conditions (CK) without MeOH treatment for 7 days.
(C) Chlorophyll content of the wild-type and tsd2 mutants in the dark-induced leaf senescence assay under treatment of 1% MeOH (MeOH) or control conditions (CK) without MeOH treatment. In (B and C), the apical 10 cm of the second upper leaves from 6-week-old plants was used for analysis.
(D) Expression level of JA biosynthetic and regulating genes in the wild-type, PMEI-1oe, and PME11oe plants. RNA samples were collected from the second upper leaves from 2-month-old plants.
(E) The relative expression level of JA biosynthesis genes under treatment with 0.1% MeOH, 1% MeOH, or without MeOH treatment (CK). Leaves from 2-month-old wild-type plants were used for the expression analysis.
(F) The content of JA-Ile in the second upper leaves of the wild-type and tsd2 mutants under treatment of 1% MeOH (MeOH) or control conditions (CK) without MeOH treatment for 3 days. Six-week-old plants under hydroponic culture were used for analysis.
(G) Exogenous JA induced leaf senescence in both the wild-type and tsd2-1 plants. Leaves used in the dark-induced senescence assay were collected from 2-month-old plants and treated with or without 3 μM MeJA for 4 days.
(H) Chlorophyll content analysis. Values are expressed as the mean ± SD of five measurements.
In (A, C–F, and H), data are shown as mean ± SD. Three biological repeats were performed and asterisks indicate the levels of statistical significance as determined by Student's t test: *P < 0.05; **P < FW, fresh weight; WT, wild-type.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

8. Figure 7
OsSRT1 Is Involved in the Regulation of MeOH-Jasmonate Cascade Genes and Leaf Senescence.
(A) SRT1.RNAi plants exhibit accelerated leaf senescence at 10 weeks old. Wild-type and SRT1.RNAi plants were grown in the greenhouse and managed following essentially normal agricultural practice.
(B and C) Relative expression of OsSRT1 (B) and H3K9 acetylation level (C) upon leaf senescence. Flag leaves from wild-type plants were used for analysis. DAF, day after flowering.
(D and E) Relative expression of OsPME1 (D) and JA biosynthetic genes (E) in the wild-type and SRT1.RNAi plants. Flag leaves from 2-month-old wild-type and SRT1.RNAi plants were used for analysis.
(F and G) Content of MeOH (F) and JA-Ile (G) in the wild-type and SRT1.RNAi plants. Flag leaves from 2-month-old wild-type and SRT1.RNAi plants were used for analysis.
In (B and D–G), data are shown as means ± SD. Three biological repeats were performed and asterisks indicate the levels of statistical significance as determined by Student's t test: *P < 0.05; **P < FW, fresh weight; WT, wild-type.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

9. Figure 8
Epigenetic Regulation of the MeOH-Jasmonates Cascade by OsSRT1.
(A) Schema of the promoter regions used for the ChIP analysis.
(B–E) ChIP analysis of the H3K9Ac levels of OsPME1 (B) and JA biosynthetic gene (C–E) promoters in the wild-type and SRT1.RNAi plants.
(F and G) ChIP analysis of OsSRT1 enrichment at the promoter regions of OsPME1 (F) and ACX4 (G).
Values are expressed as the mean ± SD of three biological repeats. Mean values followed by * or ** are significant at the 0.05 or 0.01 probability level, respectively. WT, wild-type.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions