Ethylene regulates post-germination seedling growth in wheat through spatial and temporal modulation of ABA/GA balance

Abstract

This study aimed to gain insights into the molecular mechanisms underlying the role of ethylene in regulating germination and seedling growth in wheat by combining pharmacological, molecular, and metabolomics approaches. Our study showed that ethylene does not affect radicle protrusion but controls post-germination endospermic starch degradation through transcriptional regulation of specific α-amylase and α-glucosidase genes, and this effect is mediated by alteration of endospermic bioactive gibberellin (GA) levels, and GA sensitivity via expression of the GA signaling gene, TaGAMYB. Our data implicated ethylene as a positive regulator of embryo axis and coleoptile growth through transcriptional regulation of specific TaEXPA genes. These effects were associated with modulation of GA levels and sensitivity, through expression of GA metabolism (TaGA20ox1, TaGA3ox2, and TaGA2ox6) and signaling (TaGAMYB) genes, respectively, and/or the abscisic acid (ABA) level and sensitivity, via expression of specific ABA metabolism (TaNCED2 or TaCYP707A1) and signaling (TaABI3) genes, respectively. Ethylene appeared to regulate the expression of TaEXPA3 and thereby root growth through its control of coleoptile ABA metabolism, and root ABA signaling via expression of TaABI3 and TaABI5. These results show that spatiotemporal modulation of ABA/GA balance mediates the role of ethylene in regulating post-germination storage starch degradation and seedling growth in wheat.

Keywords: Coleoptile; embryo axis; gene expression; germination; plant hormones; root; seedling; starch degradation.

Fig. 1.

Effects of treatment with an ethylene biosynthesis inhibitor, ethephon, gibberellin, or an abscisic acid biosynthesis inhibitor on germination and seedling growth. Germination (A), and lengths of embryo axis (B), coleoptile (C), primary root (D), and seminal root (E) in response to seed imbibition with the ET biosynthesis inhibitor, aminoethoxyvinylglycine (AVG, 1 mM), AVG (1 mM)+ethephon (10 μM), AVG (1 mM)+GA₃ (50 μM), or AVG (1 mM)+the ABA biosynthesis inhibitor, fluridone (FLU, 50 μM). Data are means ±SE, n=3, where n represents a batch of 20 seeds. Different letters indicate statistically significant differences between control and AVG-treated samples, while asterisks represent statistically significant differences between AVG treatment and treatment with AVG+ethephon, AVG+GA₃, or AVG+FLU (P<0.05; Student’s t-test). DAI, day(s) after imbibition.

Fig. 2.

Ethylene levels in different seedling tissues. ET level in the endosperm (A), embryo axis (B), coleoptile (C), and root (D) in response to seed imbibition with AVG (1 mM). Data are means ±SE, n=3, where n represents a batch of eight tissues (for endosperm, embryo axis at 3 DAI, coleoptile, or root) or a batch of 20 tissues (for embryo axis at 1 DAI). Asterisks indicate statistically significant differences in ethylene levels between control and AVG-treated samples (P<0.05; Student’s t-test). DAI, day(s) after imbibition.

Fig. 3.

Expression patterns of starch-degrading genes in the endosperm during imbibition. Relative transcript levels of TaAMY (A–C), TaBAM (D–H), and TaAGL (I, J) genes in AVG-treated samples and their respective controls. Transcript levels of TaAMY, TaBAM, and TaAGL genes were determined using Taβ-actin as a reference gene and expressed relative to the transcript levels of TaAMY1 in the control endosperm at 3 DAI, and the transcript levels of TaBAM1 and TaAGL1 in the control endosperm at 0 DAI, respectively, which were set to a value of 1. Data are means of three biological replicates ±SE. Asterisks indicate statistically significant differences in expression levels between control and AVG-treated samples (P<0.05; Student’s t-test). DAI, day(s) after imbibition.

Fig. 4.

Activities of starch-degrading enzymes in the endosperm during imbibition. Activities of α-amylase (A), β-amylase (B), and α-glucosidase (C) in AVG-treated samples and their respective controls. Data are means of three biological replicates ±SE. Asterisks indicate statistically significant differences in enzyme activities between control and AVG-treated samples (P<0.05; Student’s t-test). DAI, day(s) after imbibition.

Fig. 5.

Endosperm dry weight and starch and soluble sugar contents during imbibition. Dry weight (A), and contents of starch (B), sucrose (C), maltose (D), glucose (E), and fructose (F) in AVG-treated samples and their respective controls. Data are means of three biological replicates ±SE. Asterisks indicate statistically significant differences in dry weight and starch and soluble sugar contents between control and AVG-treated samples (P<0.05; Student’s t-test). DAI, day(s) after imbibition.

Fig. 6.

Expression patterns of gibberellin and abscisic acid metabolism genes, and endogenous gibberellin and abscisic acid levels in the endosperm during imbibition. Relative transcript levels of TaGA20ox1 (A), TaGA3ox2 (B), TaGA2ox6 (C), TaNCED2 (F), and TaCYP707A genes (G and H) in AVG-treated samples and their respective controls. Transcript levels were determined exactly as described in Fig. 3 and expressed relative to the transcript levels of TaGA20ox1, TaGA3ox2, TaGA2ox6, TaNCED2, and TaCYP707A1 in the control endosperm at 0 DAI, which were set to a value of 1. Endospermic GA₁ (D), GA₄ (E), and ABA (I) levels in AVG-treated samples and their respective controls. Data are means of three biological replicates ±SE except that the GA₁ data for the control samples at 5 DAI are means of five biological replicates ±SE. Asterisks indicate statistically significant differences in expression levels or hormone contents between control and AVG-treated samples (P<0.05; Student’s t-test). DAI, day(s) after imbibition.

Fig. 7.

Endospermic gibberellin and abscisic acid levels in response to embryo excision before the start of imbibition. Endospermic GA₁ (A), GA₄ (B), and ABA (C) levels of seeds imbibed for 5 d and 7 d with embryo axis (control) and no embryo axis (embryo excised). Data are means of three biological replicates ±SE. Asterisks indicate statistically significant differences in hormone contents between endosperm samples imbibed with and with no embryo (P<0.05; Student’s t-test). n.d., not detected.

Fig. 8.

Expression patterns of gibberellin and abscisic acid signaling genes in the endosperm during imbibition. Transcript levels of TaRHT1 (A), TaGAMYB (B), TaABI3 (C), and TaABI5 (D) were determined in AVG-treated samples and their respective controls exactly as described in Fig. 3 and expressed relative to their respective transcript levels in the control endosperm at 0 DAI, which were set to a value of 1. Data descriptions are as shown in Fig. 3. DAI, day(s) after imbibition.

Fig. 9.

Expression patterns of gibberellin metabolism genes in the embryo axis, coleoptile, and root of post-germination seedlings. Relative transcript levels of TaGA20ox1 (A, D, and G), TaGA3ox2 (B, E, and H), and TaGA2ox6 (C, F, and I) in AVG-treated samples and their respective controls. Transcript levels of each gene were determined exactly as described in Fig. 3 and expressed relative to their respective transcript level in the control embryo axis at 1 DAI, which was set to a value of 1. Data descriptions are as shown in Fig. 3. DAI, day(s) after imbibition; n.d., not detected.

Fig. 10.

Gibberellin and abscisic acid levels in the embryo axis, coleoptile, and root of post-germination seedlings. GA₁ (A, D, and G), GA₄ (B, E, and H), and ABA (C, F, and I) in AVG-treated samples and their respective controls. Data are means of three biological replicates ±SE. Asterisks indicate statistically significant differences in hormone contents between control and AVG-treated samples (P<0.05; Student’s t-test). DAI, day(s) after imbibition; n.d., not detected.

Fig. 11.

Expression patterns of gibberellin and abscisic acid signaling genes in the embryo axis, coleoptile, and root of post-germination seedlings. Transcript levels of TaRHT1 (A, E, and I), TaGAMYB (B, F, and J), TaABI3 (C, G, and K), and TaABI5 (D, H, and L) were determined in AVG-treated samples and their respective controls exactly as described in Fig. 3 and expressed relative to their respective transcript levels in the control embryo axis at 1 DAI, which were set to a value of 1. Data descriptions are as shown in Fig. 3. DAI, day(s) after imbibition; n.d., not detected.

Fig. 12.

Expression patterns of abscisic acid metabolism genes in the embryo axis, coleoptile, and root of post-germination seedlings. Relative transcript levels of TaNCED1 (A, E, and I), TaNCED2 (B, F, and J), TaCYP707A1 (C, G, and K), and TaCYP707A2 (D, H, and L) in AVG-treated samples and their respective controls. Transcript levels of NCED and CYP707A genes were determined exactly as described in Fig. 3 and expressed relative to TaNCED1 and TaCYP707A1 transcript levels in the control embryo axis at 1 DAI, respectively, which were set to a value of 1. Data descriptions are as shown in Fig. 3. DAI, day(s) after imbibition; n.d., not detected.

Fig. 13.

Expression patterns of α-expansin genes in the embryo axis, coleoptile, and root of post-germination seedlings. Transcript levels of TaEXPA3 (A, D, and G), TaEXPA7 (B, E, and H), and TaEXPA9 (C, F, and I) were determined in AVG-treated samples and their respective controls exactly as described in Fig. 3 and expressed relative to the transcript level of TaEXPA3 in the control embryo axis at 1 DAI, which was set to a value of 1. Data descriptions are as shown in Fig. 3. DAI, day(s) after imbibition.

Fig. 14.

Schematic depiction of the role of ethylene (ET) in regulating abscisic acid (ABA)/gibberellin (GA) balance and seedling growth in wheat. ET enhances seedling growth by inducing endospermic and non-endospermic (embryo axis, coleoptile, and root) GA level and sensitivity through expression of GA biosynthetic (TaGA20ox1 and TaGA3ox2) and/or catabolic (TaGA2ox6), and GA signaling (TaGAMYB) genes. Endospermic bioactive GA, which mainly comprises GA transported from non-endospermic tissues, induces storage starch degradation through enhancing the expression levels of specific α-amylase (TaAMY1, TaAMY3, and TaAMY4) and α-glucosidase (TaAGL1) genes and the activity of α-amylase and α-glucosidase, while GAs in non-endospermic tissues induce cell wall expansion via expression of specific α-expansin (TaEXPA3, and/or TaEXPA7, and/or TaEXPA9) genes. Furthermore, ET represses the ABA level and signaling in non-endospermic tissues via expression of ABA biosynthetic (TaNCED2), catabolic (TaCYP707A1), and ABA signaling (TaABI3 and/or TaABI5) genes, contributing to the induction cell wall expansion via expression of TaEXPA genes. ET does not affect radicle protrusion in wheat seeds.