Volume 7, Issue 5, Pages 841-855 (May 2014) Short-Term and Continuing Stresses Differentially Interplay with Multiple Hormones to Regulate Plant Survival and Growth  Yang Cangjing , Liu Jingjing , Dong Xinran , Cai Zhenying , Tian Weidong , Wang Xuelu   Molecular Plant  Volume 7, Issue 5, Pages 841-855 (May 2014) DOI: 10.1093/mp/ssu013 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Differentially Expressed Genes under Short-Term and Sustained ABA Treatments. (A) Venn diagrams show the number of differentially expressed genes between sustained and short-term ABA treated samples. Differentially expressed genes were defined by p-value < 0.05 and fold change > 1.4 between ABA and mock treatments. Global coverage for specific subgroup genes is indicated in brackets. (B) Illustration of subgroup genes regulated by short-term and sustained ABA treatments. Molecular Plant 2014 7, 841-855DOI: (10.1093/mp/ssu013) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 Short-Term ABA, but Not Sustained ABA, Down-Regulates Expression of Genes Related to BR Signaling. (A) Histograms show percentage of the BR-responsive genes regulated by short-term or sustained ABA treatments. The x-axis shows the log2 expression levels of genes upon the indicated ABA treatments. Numbers in boxes indicate percentage sum of up-regulated (slash) and down-regulated (gray) genes. p-values were computed with using the Fisher’s exact test. ‘short_up’ and ‘short_down’ indicate putative BR-up-regulated and BR-down-regulated genes by short-term ABA, respectively. ‘long_up’ and ‘long_down’ indicate putative BR-up-regulated and BR-down-regulated genes by sustained ABA, respectively. ‘short_up’, ‘short_down’, ‘long_up’, and ‘long_down’ were also used in Figures 3A, 4A, 5A, 6A, and 7A. (B) Relative expression of BR-responsive genes, Saur-AC1 (AT4G38850), DWF4 (AT3G50660), and CPD (AT5G05690), by indicated treatments. Error bars are standard errors (SE). SE was calculated based on the data of three independent experiments. Significant levels of difference from mock treatments were determined by Student’s t-test. * p-value < 0.05; ** p-value < 0.01. The SE calculation and significant levels were also used in Figures 2D, 3B, 4B, 4D, 5B, 5D, 6B, 6D, and 7B. (C) Heat map shows regulation of genes involved in BR biosynthesis, metabolism, positive signaling components, and negative signaling components by short-term (Short) and sustained (Long) ABA treatments. (D) Relative expression levels of Saur-AC1, DWF4, and CPD regulated by short-term ABA compared to mock treatment in Col-0, det2-1, bri1-301, and BES1-RNAi backgrounds. Molecular Plant 2014 7, 841-855DOI: (10.1093/mp/ssu013) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 Auxin Signaling May Be Accelerated by Short-Term ABA and Sustained ABA at Different Levels. (A) Histograms show the percentage of auxin-responsive genes regulated by short-term or sustained ABA treatment. The x-axis shows the log2 gene expression levels upon the indicated ABA treatment. Numbers in boxes indicate the sum percentage of up-regulated (slash) and down-regulated (gray). p-value was computed with the Fisher’s exact test. (B) Relative expression of auxin-responsive genes, GH3-1 (AT2G14960), GH3-6 (AT5G54510), and IAA30 (AT3G62100), which are all up-regulated by auxin signal. (C) Heat map shows the regulation of genes involved in auxin biosynthesis, metabolism, positive signaling component, and negative signaling components by short-term (Short) and sustained (Long) ABA treatments. Molecular Plant 2014 7, 841-855DOI: (10.1093/mp/ssu013) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 GA Signaling Is Repressed by Short-Term and Sustained ABA Likely at Different Levels. (A) Histograms show percentage of GA-responsive genes regulated by short-term or sustained ABA treatment. The x-axis shows the log2 gene expression levels upon the indicated ABA treatment. Numbers in boxes indicate the sum percentage of up-regulated (slash) and down-regulated (gray). p-value was computed with the Fisher’s exact test. (B) Relative expression of GA-responsive genes, GA3ox1 (AT1G15550), GA20ox2 (AT5G51810), and SCL3 (AT1G50420), which are all up-regulated by GA signal. (C) Heat map shows the regulation of genes involved in GA biosynthesis, metabolism, positive signaling components, and negative signaling components by short-term (Short) and sustained (Long) ABA treatments. (D) Relative expression levels of GA3ox1, GA20ox2, and SCL3 regulated by short-term and sustained ABA as compared with mock treatment in Col-0 and gid1a-1gid1c-1 background. Molecular Plant 2014 7, 841-855DOI: (10.1093/mp/ssu013) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 Sustained ABA, but Not Short-Term ABA, May Repress CK Signaling. (A) Histograms show the percentage of CK-responsive genes regulated by short-term or sustained ABA treatment. The x-axis shows the log2 gene expression levels upon the indicated ABA treatment. Numbers in boxes indicate the sum percentage of up-regulated (slash) and down-regulated (gray). p-value was computed with the Fisher’s exact test. (B) Relative expression of CK-responsive genes, ARR6 (AT5G62920), ARR9 (AT3G57040), and CRF3 (AT5G53290), which are all up-regulated by CK signal. (C) Heat map shows the regulation of genes involved in CK biosynthesis, metabolism, positive signaling component, and negative signaling components by short-term (Short) and sustained (Long) ABA treatments. (D) Relative expression levels of ARR6, ARR9, and CRF3 regulated by short-term and sustained ABA compared with mock treatment in Col-0, ahk2cre1, and ahk3cre1 background. Molecular Plant 2014 7, 841-855DOI: (10.1093/mp/ssu013) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 Short-Term ABA, but Not Sustained ABA, Likely Promotes Ethylene Signaling mainly through Up-Regulating Genes Encoding Ethylene Signaling Components. (A) Histograms show the percentage of ethylene-responsive genes regulated by short-term or sustained ABA treatment. The x-axis shows the log2 gene expression levels upon the indicated ABA treatment. Numbers in boxes indicate the sum percentage of up-regulated (slash) and down-regulated (gray). p-value was computed with the Fisher’s exact test. (B) Relative expression of ethylene-responsive genes, ERF1 (AT3G23240), EDF1 (AT1G25560), and EDF4 (AT1G13260), which are all up-regulated by ethylene signal. (C) Heat map shows the regulation of genes involved in ethylene biosynthesis, metabolism, positive signaling component, and negative signaling components by short-term (Short) and sustained (Long) ABA treatments. (D) Relative expression levels of ERF1, EDF1, and EDF4 regulated by short-term ABA treatment compared with mock treatment in Col-0, EIN3-OX, and ein3-1 background. Molecular Plant 2014 7, 841-855DOI: (10.1093/mp/ssu013) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 7 Short-Term ABA, but Not Sustained ABA, Depresses JA Signal. (A) Histograms show the percentage of JA-responsive genes regulated by short-term or sustained ABA treatment. The x-axis shows the log2 gene expression levels upon the indicated ABA treatment. Numbers in boxes indicate the sum percentage of up-regulated (slash) and down-regulated (gray). p-value was computed with the Fisher’s exact test. (B) Relative expression of JA-responsive genes, LOX3 (AT1G17420), LOX4 (AT1G72520), and OPR3 (AT2G06050), which are all up-regulated by JA signal. (C) Heat map shows the regulation of genes involved in JA biosynthesis, metabolism, positive signaling component, and negative signaling components by short-term (Short) and sustained (Long) ABA treatments. (D) Regulation of JA signaling by short-term ABA treatment. Short-term ABA impairs JA signaling at the protein level and inhibits many JAZ family genes’ expression through a negative feedback regulation. Molecular Plant 2014 7, 841-855DOI: (10.1093/mp/ssu013) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 8 A Proposed Model to Illustrate how Short-Term and Sustained ABA Treatments Integrate with Other Hormones to Regulate Plant Survival and Growth under Short-Term Stress (Response Phase) and Continuing Stress (Restitution Phase). Short-term ABA likely represses BR, JA, and GA signaling at the protein level, and promotes auxin signaling at the protein level and ethylene signaling at the RNA level. Sustained ABA may repress CK signaling at the protein level, and inhibit GA signaling and promote auxin signaling at the RNA level. BES1, EIN3, GID1a/GID1c, and AHK2/AHK3/CRE1 are required for the effect of short-term or sustained ABA on the signaling outputs of BR, ethylene, GA, and CK, respectively. Molecular Plant 2014 7, 841-855DOI: (10.1093/mp/ssu013) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions