Figure 1.
Effect of after-ripening on the release of dormancy in wheat seeds.
Germination percentage of dormant (D) and after-ripened (AR) seeds imbibed in water (D and AR) and ABA (AR) over 4 days (A). Comparison of D and AR seeds following 4 day imbibition in water (D and AR) and ABA (AR). DAI, days after imbibition.
Figure 2.
Changes in the expression of brassinosteroid (BR) metabolism and signaling genes in response to after-ripening.
Simplified BR metabolic and signaling pathway in plants (A). Fold changes (log2-scale) in the expression of BR metabolism (B) and signaling (C) related probesets between after-ripened (AR) and dormant (D) seeds in both dry (AR-0/D-0) and imbibed states (AR-12/D-12 and AR-24/D-24), and between AR seeds imbibed without and with ABA (AR-24/AR-24+ABA). The log2 fold change values are shown by the negative and positive numbers on the bar, and the color scale shows upregulation (red) and downregulation (olive green) of the respective probesets. Fold changes (linear-scale) in expression and the associated P values are presented in Table S2. Data are means of 3 independent biological replicates. Abbreviations: DET, de-etiolated; DWF4, dwarf 4; CPD, constitutive photomorphogenic dwarf; ROT3, rotundfolia 3; RAV, related to ABI3/VP1; BRI, brassinosteroid insensitive; BAK, BRI1-associated receptor kinase; BSK, BR-signaling kinases; BSU, BRI suppressor; BIN, brassinosteroid insensitive; BZR, brassinazole-resistant.
Figure 3.
Comparison of the expression (log2-scale) of brassinosteroid (BR) responsive genes.
Relative transcript abundance of paclobutrazol-resistance (PRE) (A, B) and brassinosteroid enhanced expression (BEE) (C, D) in dormant (D) and after-ripened (AR) seeds before imbibition (D-0 and AR-0) and during imbibition in water (D-12, AR-12, D-24, and AR-24) and ABA (AR-24+ABA). Transcript abundance was expressed relative to that in D-0 seeds, which was arbitrarily set to a value of 1. Data are means of 3 independent biological replicates ± SE. Within each imbibition time point, different letters indicate significant difference in transcript abundance between seed samples at P≤0.05.
Figure 4.
Changes in the expression of ethylene (ET) metabolism and signaling genes in response to after-ripening.
Simplified ET metabolic and signaling pathway in plants (A). Fold changes (log2-scale) in the expression of ET metabolism (B) and signaling (C) related probesets between after-ripened (AR) and dormant (D) seeds in both dry (AR-0/D-0) and imbibed states (AR-12/D-12 and AR-24/D-24), and between AR seeds imbibed without and with ABA (AR-24/AR-24+ABA). The log2 fold change values are shown by the negative and positive numbers on the bar, and the color scale shows upregulation (red) and downregulation (olive green) of the respective probesets. Fold changes (linear-scale) in expression and the associated P values are presented in Table S2. Data are means of 3 independent biological replicates. Abbreviations: ACS, 1-aminocyclopropane-1-carboxylic acid synthase; ACO, 1-aminocyclopropane-1-carboxylic acid oxidase; RAN, responsive-to-antagonist; RTE, reversion-to-ethylene sensitivity; ETR, ethylene response; ERS, ethylene response sensor; CTR, constitutive triple response; TPR, tetratricopeptide repeat; EIN2, ethylene insensitive 2; ECIP, EIN2 c-terminus interacting protein; EIN5, ethylene insensitive5; EIN3, ethylene insensitive 3; EBF, EIN3-binding F box protein; EER, enhanced ethylene response; ERF, ethylene-responsive element binding factor; MPK3, mitogen-activated protein kinase 3; MPK6, mitogen-activated protein kinase 6.
Figure 5.
Changes in the expression of cytokinin (CK) metabolism and signaling genes in response to after-ripening.
Simplified CK metabolic and signaling pathway in plants (A). Fold changes (log2-scale) in the expression of CK metabolism (B) and signaling (C) related probesets between after-ripened (AR) and dormant (D) seeds in both dry (AR-0/D-0) and imbibed states (AR-12/D-12 and AR-24/D-24), and between AR seeds imbibed without and with ABA (AR-24/AR-24+ABA). The log2 fold change values are shown by the negative and positive numbers on the bar, and the color scale shows upregulation (red) and downregulation (olive green) of the respective probesets. Fold changes (linear-scale) in expression and the associated P values are presented in Table S2. Data are means of 3 independent biological replicates. Abbreviations: DMAPP, dimethylallyl diphosphate; AMP, adenosine monophosphate; IPT, isopentenyltransferase; LOG, lonely guy; CKX, cytokinin oxidase; cZOG, cis zeatin-o-glucoside; GLU, glucosidase; AHK, Arabidopsis histidine kinase; AHP, Arabidopsis histidine-containing phosphotransmitter; ARR, Arabidopsis response regulator.
Figure 6.
Changes in expression of salicylic acid (SA) metabolism and signaling genes in response to after-ripening.
Simplified SA metabolic and signaling pathway in plants (A). Fold changes (log2-scale) in the expression of SA metabolism (B) and signaling (C) related probesets between after-ripened (AR) and dormant (D) seeds in both dry (AR-0/D-0) and imbibed states (AR-12/D-12 and AR-24/D-24), and between AR seeds imbibed without and with ABA (AR-24/AR-24+ABA). The log2 fold change values are shown by the negative and positive numbers on the bar, and the color scale shows upregulation (red) and downregulation (olive green) of the respective probesets. Fold changes (linear-scale) in expression and the associated P values are presented in Table S2. Data are means of 3 independent biological replicates. Abbreviations: ICS, isochorismate synthase; PAL, phenylalanine ammonia-lyase; GH3, glycoside hydrolase 3; SSI2, suppressor of SA insensitive 2; TRX-H5, thioredoxin-h5; TRX-H3, thioredoxin-h3; MOS3, modifier of SNC1, 3; MOS6, modifier of SNC1, 6; NPR, Arabidopsis non-expressor of pathogenesis-related genes; TGA, TGACG motif-binding factor; SNI, suppressor of NPR1-1 inducible; WHY1, whirly 1; WRKY, WRKY DNA-binding protein.
Figure 7.
Validation of the microarray data with real-time qRT-PCR.
Comparison of the microarray and qPCR data for selected probestes representing genes related to brassinosteroid (A-D), ethylene (E, F), cytokinin (G-I) and salicylic acid (J) using real time quantitative RT-PCR. Green curves in each graph represent DNA microarray data (left y-axis) while the red curves represent qPCR data (right y-axis) for both dormant (D) and after-ripened (AR) samples before imbibition (D-0 and AR-0), and after imbibition in water (D-12, D-24, AR-12 and AR-24) and ABA (AR-24+ABA). Log2 signal intensities for each probeset in both microarray and qPCR experiments were expressed relative to that derived from D-0 sample, which was arbirarily set to a value of 0. Data are means of 3 independent biological (and two technical) replicates ± SE. The probeset ID, the corresponding gene name and the pearson correlation coefficient (R) between the microarray and qPCR data are indicated at the top of each graph. Abbreviations: DET, de-etiolated; DWF4, dwarf4; BSK, BR-signaling kinase; MFT, mother of FT AND TFL1; ACO, 1-aminocyclopropane-1-carboxylic acid oxidase 1; cZOG, cis zeatin-o-glucoside; GLU, glucosidase; ARR, Arabidopsis response regulator; SSI2, suppressor of SA insensitive 2.
Figure 8.
Heat map visualization of hierarchical clustering of hormone related genes.
Expression values in log2 fold changes of genes differentially regulated (≥1-fold on log2-scale [≥2-fold on linear-scale], P≤0.05) between dormant (D) and after-ripened (AR) seeds in dry (AR-0/D-0) and hydrated (AR-12/D-12, AR24/D-24) states were used to cluster the genes on the basis of their expression pattern. The log2 fold change values are shown by the negative and positive numbers on the bar and the color scale shows upregulation (red) and downregulation (blue) of the respective probesets. Fold changes (linear-scale) in expression and the associated P values are presented in Table S3. Data are means of 3 independent biological replicates. Abbreviations: ABI5, ABA insensitive 5; ABF, ABA responsive elements-binding factor; OPR1, 12-oxophytodienoate reductase 1; AOS, allene oxide synthase; GA20OX1, GA 20 oxidase 1; GID1, GA insensitive dwarf 1; AIP2, ABI3-interacting protein 2; KAT, 3-ketoacyl-COA thiolase; LOX, lipoxygenase; LPP, lipid phosphate phosphatase; MPK, mitogen-activated protein kinase; PYL, pyrabactin resistance-like; RUB, related to ubiquitin; SnRK, SNF1-related protein kinase; ZEP, zeaxanthin epoxidase. Please see the legends of Figures 2, 4, 5 and 6 for the other gene abbreviations.