Citrus ACC synthase CiACS4 regulates plant height by inhibiting gibberellin biosynthesis

Abstract

Dwarfism is an agronomic trait that has substantial effects on crop yield, lodging resistance, planting density, and a high harvest index. Ethylene plays an important role in plant growth and development, including the determination of plant height. However, the mechanism by which ethylene regulates plant height, especially in woody plants, remains unclear. In this study, a 1-aminocyclopropane-1-carboxylic acid synthase (ACC) gene (ACS), which is involved in ethylene biosynthesis, was isolated from lemon (Citrus limon L. Burm) and named CiACS4. Overexpression of CiACS4 resulted in a dwarf phenotype in Nicotiana tabacum and lemon and increased ethylene release and decreased gibberellin (GA) content in transgenic plants. Inhibition of CiACS4 expression in transgenic citrus significantly increased plant height compared with the controls. Yeast two-hybrid assays revealed that CiACS4 interacted with an ethylene response factor (ERF), CiERF3. Further experiments revealed that the CiACS4-CiERF3 complex can bind to the promoters of 2 citrus GA20-oxidase genes, CiGA20ox1 and CiGA20ox2, and suppress their expression. In addition, another ERF transcription factor, CiERF023, identified using yeast one-hybrid assays, promoted CiACS4 expression by binding to its promoter. Overexpression of CiERF023 in N. tabacum caused a dwarfing phenotype. CiACS4, CiERF3, and CiERF023 expression was inhibited and induced by GA3 and ACC treatments, respectively. These results suggest that the CiACS4-CiERF3 complex may be involved in the regulation of plant height by regulating CiGA20ox1 and CiGA20ox2 expression levels in citrus.

Figure 1.

Expression, bioinformatics, and subcellular localization analyses of CiACS4. A) Lemon shoots from different developmental stages: S1 indicates freshly sprouted lemon shoots, S6 indicates mature shoots, S2 to S5 indicate different stages of shoot development. Scale bar = 5 cm. B) Expression analyses of CiACS4 at different developmental stages of shoots. Values are represented as the means ± standard errors (SEs) (n = 3). C) Different parts of mature shoots for CiACS4 expression analyses. Scale bar = 5 cm. D) Expression analyses of CiACS4 in different tissues of lemon. St1, St2, and St3 indicate different parts of the mature shoots. LB indicates the lateral bud. SAM indicates the shoot apical meristem. Values are represented as the means ± standard errors (SEs) (n = 3). E) Protein alignment of CiACS4 and its homologous proteins from Arabidopsis and rice. F) Phylogenetic tree from different homologous sequences of CiACS4. Neighbor-joining phylogenetic tree was constructed using MEGA6 software with 1,000 bootstrap replicates. The circle indicates CiACS4. Scale bar represents 20 different for every 100 amino acids. G) Subcellular localization of the CiACS4-GFP fusion protein in the epidermal cells of N. benthamiana leaves. GFP, bright field. Merged images are shown. Red color indicates the fluorescence of the nuclear marker (VirD2NLS-mCherry). Scale bar = 25 μm.

Figure 2.

Physiological characterization of 35S:CiACS4 transgenic lemon. A) Phenotypic analyses of 35S:CiACS4 transgenic lemon. Scale bar = 5 cm. B) Comparison analyses of the apical meristem of 35S:CiACS4 transgenic lemon and the controls. Scale bar = 1 cm. C) Comparison analyses of the internode between 35S:CiACS4 transgenic lemon and the controls. Scale bar = 0.5 cm. D–F) Plant height D), internode length E), and leaf number F) analyses of 35S:CiACS4 transgenic lemon (5-month-old plants were used). Values are represented as the means ± standard errors (SEs) (n = 7). Asterisks indicate the significant differences: **P < 0.01 and *P < 0.05, Student's t-test. G) Cross sections of the internode from the controls and 35S:CiACS4 transgenic lemon. H) Cell numbers based on the cross sections of the controls and 35S:CiACS4 transgenic lemon. Values are represented as the means ± SEs (n = 15). Asterisks indicate the significant differences: **P < 0.01, Student's t-test. I) Longitudinal sections of the internode from the controls and 35S:CiACS4 transgenic lemon. Scale bar = 50 μm. J) Cell diameter based on the longitudinal sections of the controls and 35S:CiACS4 transgenic lemon. Values are represented as the means ± SEs (n = 15). Asterisks indicate the significant differences: **P < 0.01, Student's t test. OE#13 and OE#23 represent 2 35S:CiACS4 transgenic lemon lines.

Figure 3.

Physiological characterization of RNAi-CiACS4 transgenic lemon. A) Phenotypic characterization of RNAi-CiACS4 transgenic lemon. Scale bar = 5 cm. B) Comparison analyses of apical meristem of RNAi-CiACS4 transgenic lemon and the controls. Scale bar = 1 cm. C) Comparison analyses of the internodes of RNAi-CiACS4 transgenic lemon and the controls. Scale bar = 0.5 cm. D–F) Analyses of plant height D), internode length E), and number of leaves F) in RNAi-CiACS4 transgenic lemon (5-month-old plants were used). Values are represented as the means ± SEs (n = 6). Asterisks indicate the significant differences: **P < 0.01 and *P < 0.05, Student's t-test. G) Cross sections of the internode from the controls and RNAi-CiACS4 transgenic lemon. H) Cell numbers based on the cross sections of the controls and RNAi-CiACS4 transgenic lemon. Values are represented as the means ± SEs (n = 15). Asterisks indicate the significant differences: **P < 0.01, Student's t-test. I) Longitudinal sections of the internode from the controls and RNAi-CiACS4 transgenic lemon. Scale bar = 50 μm. J) Cell diameter based on the longitudinal sections of the controls and RNAi-CiACS4 transgenic lemon. Values are represented as the means ± SEs (n = 15). RNAi#15 and RNAi#25 represent 2 RNAi-CiACS4 transgenic lemon lines. Asterisks indicate the significant differences: **P < 0.01, *P < 0.05. NS > 0.05, Student's t-test.

Figure 4.

Ethylene and GA participate in CiACS4 regulation of plant height. A–D) Levels of Gibberellin A3 (GA₃) A), Gibberellin A20 (GA₂₀) B), indole-3-acetic acid (IAA) C), and ethylene D) in 35S:CiACS4 and RNAi-CiACS4 transgenic lemon. OE#13 and OE#23 represent 2 35S:CiACS4 transgenic lemon lines. RNAi#15 and RNAi#25 represent 2 RNAi-CiACS4 transgenic lemon lines. Values are represented as the means ± SEs (n = 3). Asterisks indicate the significant differences: **P < 0.01 and *P < 0.05, Student's t-test. E) Morphological analyses of 2-month-old lemon seedlings treated with Gibberellin A3 (GA₃), brassinolide (BL), and indole-3-acetic acid (IAA). Scale bar = 5 cm. F) Analyses of the % increase in plant height after treatment of WT lemon under different treatments. Values are represented as the means ± SEs (n ≥ 5). Asterisks indicate the significant differences: **P < 0.01 and *P < 0.05, Student's t-test.

Figure 5.

Morphological characterization of treated lemon seedlings. A) Phenotype of treated lemon seedlings. Two-month-old lemon seedlings were treated with ACC, GA₃, and ACC + GA₃. Scale bar = 5 cm. B) Analyses of the % increase in plant height after treatment of wild-type lemon under different treatments. Values are represented as the means ± SEs (n ≥ 7). Asterisks indicate the significant differences: *P < 0.05 and **P < 0.01, Student's t-test. C) Cell numbers based on the cross sections of WT lemon with different treatments. Values are represented as the means ± SEs (n = 15). Asterisks indicate the significant differences: *P < 0.05 and **P < 0.01, Student's t-test. D) Cell diameter based on the longitudinal sections of WT lemon with different treatments. Values are represented as the means ± SEs (n = 15). Asterisks indicate the significant differences: *P < 0.05 and **P < 0.01, Student's t test. E) Expression levels of CiACS4 in plants treated with ACC, GA₃, and ACC + GA₃. Values are represented as the means ± SEs (n = 3). Asterisks indicate the significant differences: **P < 0.01, Student's t-test. F) Morphological analyses of 35S:CiACS4 transgenic lemon after treatment with ACC, GA₃, and ACC + GA₃. G) Analyses of the % increase in plant height of 35S:CiACS4 transgenic lemon after treatment with ACC, GA₃, and ACC + GA₃. Values are represented as the means ± SEs (n ≥ 6). Asterisks indicate the significant differences: *P < 0.05 and **P < 0.01, Student's t-test. H) Morphological analyses of RNAi-CiACS4 transgenic lemon after treatment with ACC, GA₃, and ACC + GA₃. I) Analyses of the % increase in plant height of RNAi-CiACS4 transgenic lemon after treatment with ACC, GA₃, and ACC + GA₃. Values are represented as the means ± SEs (n ≥ 6). Asterisks indicate the significant differences: *P < 0.05 and **P < 0.01, Student's t-test.

Figure 6.

CiERF3 protein interacts with CiACS4. A) Yeast two-hybrid assays confirmed the interaction of CiACS4 and CiERF3. B) Interaction of CiACS4 and CiERF3 was confirmed by BiFC. YFP^N-CiACS4 and YFP^C-CiERF3 interacted to form a functional YFP in N. benthamiana cells. YFP^N + YFP^C-CiERF3 and YFP^N-CiACS4 + YFP^C were used as the negative controls. Red color indicates the fluorescence of nuclear marker (VirD2NLS-mCherry). Scale bar = 25 µm. C) Pull-down assays confirmed the interaction between CiACS4 with CiERF3. D) Expression levels of CiGA3ox1 in 35S:CiACS4 and RNAi-CiACS4 transgenic lemon. Values are represented as the means ± SEs (n = 3). Asterisks indicate the significant differences: **P < 0.01, Student's t-test. E–F) Expression levels of CiGA20ox1E) and CiGA20ox2F) in 35S:CiACS4 and RNAi-CiACS4 transgenic lemon. OE#13 and OE#23 represent 2 35S:CiACS4 transgenic lemon lines. RNAi#15 and RNAi#25 represent 2 RNAi-CiACS4 transgenic lemon lines. Values are represented as the means ± SEs (n = 3). Asterisks indicate the significant differences: **P < 0.01, Student's t-test.

Figure 7.

CiERF3 binding to the promoter of CiGA20ox1 and CiGA20ox2. A) Transcriptional activity of CiERF3. P53 indicates pGBKT7 + p53; BD-empty indicates pGBKT7; BD-CiERF3 indicates pGBKT7-CiERF3. B) CiERF3 binding to CiGA20ox1 and CiGA20ox2 promoters was confirmed by Y1H assays. AD indicates pGADT7 + pAbAi-CiGA20ox1 or pAbAi-CiGA20ox2; CiERF3 indicates pGADT7-CiERF023 + pAbAi-CiGA20ox1 or pAbAi-CiGA20ox2. C–D) CiERF3 binding to CiGA20ox1C) and CiGA20ox2D) promoters was confirmed via EMSAs. + and − indicate the presence and absence of the probe or protein, respectively; 40× and 80× specify the increasing amounts of cold probe for competition. E–F CiERF3 inhibited the expression of CiGA20ox1E) and CiGA20ox2F) according to the LUC assay. Values are represented as the means ± SEs (n = 3). The diagram of vectors used for the LUC assays is shown for each. The reporter vector contained CiGA20ox1 or CiGA20ox2 promoters fused to LUC (empty vector, CiERF3). Asterisks indicate the significant differences: **P < 0.01 and *P < 0.05, Student's t-test.

Figure 8.

Interaction of the CiERF023 protein with the promoter of CiACS4. A) Subcellular localization of the CiERF023-EGFP fusion reporter in the epidermal cells of N. benthamiana leaves. GFP, bright field. Merged images are shown. RFP indicates the fluorescence of nuclear marker (VirD2NLS-mCherry). Scale bar = 25 μm. B) Transcriptional activity analyses of CiERF023. Positive indicates pGBKT7 + p53; negative indicates pGBKT7; BD-CiERF023 indicates pGBKT7-CiERF023. C) Confirmation of CiERF023 binding to the CiACS4 promoter using Y1H assay. Positive indicates pGADT7-Rec-p53 + p53-AbAi; AD indicates pGADT7 + CiACS4-AbAi; CiERF023 indicates pGADT7-CiERF023 + CiACS4pro-AbAi. D) Confirmation of CiERF23 binding to the CiACS4 promoter via EMSAs. + and − indicate the presence and absence of the probe or protein, respectively. 25×, 50×, and 100× indicate the increasing number of cold probe for competition. E) CiERF023 activated the expression of CiACS4 in the LUC assay. Values are represented as the means ± SEs (n = 3). Asterisks indicate the significant differences: **P < 0.01, Student's t-test.

Figure 9.

Morphological characterization of 35S:CiERF023 transgenic N. tabacum. A) Phenotypic characterization of 35S:CiERF023 N. tabacum. Scale bar = 5 cm. B–D) Plant height B), internode length C), and leaf number D) analyses of 35S:CiERF023 transgenic N. tabacum. Values are represented as the means ± SEs (n ≥ 12). E) Cell numbers based on the cross sections of the controls and 35S:CiACS4 transgenic lemon. Values are represented as the means ± SEs (n = 15). F) Cell diameter based on the longitudinal sections of the controls and 35S:CiACS4 transgenic lemon. Values are represented as the means ± SEs (n = 15). G) Levels of ethylene in 35S:CiERF023 transgenic N. tabacum. Values are represented as the means ± SEs (n = 3). Asterisks indicate the significant differences: **P < 0.01, Student's t-test. H) Levels of GA₃ in 35S:CiERF023 transgenic N. tabacum. Values are represented as the means ± SEs (n = 3). Asterisks indicate the significant differences: **P < 0.01 and *P < 0.05, Student's t-test. I) Levels of GA₂₀ in 35S:CiERF023 transgenic N. tabacum. Values are represented as the means ± SEs (n = 3). Asterisks indicate the significant differences: **P < 0.01 and *P < 0.05, Student's t-test. J–L) Expression levels of NtACS4J), NtGA20ox1K), and NtGA20ox2L). Values are represented as the means ± SEs (n = 3). CiERF023#2 and CiERF023#6 represent 2 35S:CiERF023 transgenic N. tabacum lines. Asterisks indicate the significant differences: **P < 0.01, Student's t-test.

Figure 10.

CiERF023 and CiERF3 antagonistically regulate the expression of CiACS4. A–B) Expression levels of CiERF3A) and CiERF023B) in lemon seedlings treated with 1-aminocyclopropane-1-carboxylic acid (ACC), Gibberellin A3 (GA₃), and ACC + GA₃. Values are represented as the means ± SEs (n = 3). Asterisks indicate the significant differences: **P < 0.01, Student's t-test. C–D) Expression levels of CiERF3C) and CiERF023D) in 35S:CiACS4 and RNAi-CiACS4 transgenic lemon. OE#13 and OE#23 represent 2 35S:CiACS4 transgenic lemon lines. RNAi#15 and RNAi#25 represent 2 RNAi-CiACS4 transgenic lemon lines. Values are represented as the means ± SEs (n = 3). Asterisks indicate the significant differences: **P < 0.01 and *P < 0.05. E) Confirmation of CiERF3 binding to the CiACS4 promoter via Y1H assay. Positive indicates pGADT7-Rec-p53 + p53-AbAi; AD indicates pGADT7 + CiACS4-AbAi. CiERF3 indicates pGADT7-CiERF3 + CiACS4pro-AbAi. F) CiERF023 activated the expression of CiACS4 in the LUC assay. Values are represented as the means ± SEs (n = 3). Asterisks indicate the significant differences: **P < 0.01, Student's t-test. G) Confirmation of CiERF023 binding to the CiERF3 promoter via Y1H assay. Positive indicates pGADT7-Rec-p53 + p53-AbAi; AD indicates pGADT7 + CiERF3-AbAi. CiERF023 indicates pGADT7-CiERF023 + CiERF3pro-AbAi. H) CiERF023 activated the expression of CiERF3 in the LUC assay. Values are represented as the means ± SEs (n = 3). Asterisks indicate the significant differences: **P < 0.01, Student's t-test. I) Working model of the CiACS4–CiERF3 complex for the regulation of plant height in citrus.