Overexpression of 9- cis-Epoxycarotenoid Dioxygenase Gene, IbNCED1, Negatively Regulates Plant Height in Transgenic Sweet Potato

Abstract

Plant height is one of the key agronomic traits for improving the yield of sweet potato. Phytohormones, especially gibberellins (GAs), are crucial to regulate plant height. The enzyme 9-cis-epoxycarotenoid dioxygenase (NCED) is the key enzyme for abscisic acid (ABA) biosynthesis signalling in higher plants. However, its role in regulating plant height has not been reported to date. Here, we cloned a new NCED gene, IbNCED1, from the sweet potato cultivar Jishu26. This gene encoded the 587-amino acid polypeptide containing an NCED superfamily domain. The expression level of IbNCED1 was highest in the stem and the old tissues in the in vitro-grown and field-grown Jishu26, respectively. The expression of IbNCED1 was induced by ABA and GA3. Overexpression of IbNCED1 promoted the accumulation of ABA and inhibited the content of active GA3 and plant height and affected the expression levels of genes involved in the GA metabolic pathway. Exogenous application of GA3 could rescue the dwarf phenotype. In conclusion, we suggest that IbNCED1 regulates plant height and development by controlling the ABA and GA signalling pathways in transgenic sweet potato.

Keywords: ABA; GA; IbNCED1; plant height; sweet potato.

Figure 1

Sequence alignment (A) and phylogenetic tree (B) of IbNCED1 with its homologs from other plants.

Figure 2

Expression analysis of IbNCED1 in different tissues of Jishu26 plants. The expression analysis of IbNCED1 in different tissues of in vitro-grown (A) and field-grown (B) plants of Jishu26. LL: Leaflet; L: Leaf; PR: Pencil root; R: Root; S: Stem; SR: Storage root; YS: Young stem. The transcript levels of IbNCED1 in the leaf tissue or leaflet were set to 1. The expression analysis of IbNCED1 in different tissues of Jishu26 plants after different time points (h) in response to 100 mM ABA (C) and 100 mM GA (D), respectively. The expression level of IbNCED1 in the plant sampled at 0 h was set to 1. The data are presented as the means ± SEs (n = 3). * indicates significant differences from that of WT at p < 0.05, according to Student’s t-test.

Figure 3

Production of the IbNCED1-overexpressing sweet potato plants. (A) Diagram of constitutive expression of the 35S promoter::IbNCED1 construct. (B) Embryogenic cultures proliferating in MS medium with 2.0 mg L⁻¹ 2,4-D. (C) Hyg-resistant calluses formedafter 4 weeks of selection on MS medium with 2.0 mg L⁻¹ 2,4-D, 100 mg L⁻¹ Carb and 10 mg L⁻¹ Hyg. (D) Germination of somatic embryos from Hyg-resistant calluses on MS medium with 1.0 mg L⁻¹ ABA and 100 mg L⁻¹ Carb. (E) Whole regenerated plantlets. (F) PCR analysis of the transgenic plants. Lane M: BL2000 plus DNA marker; Lane W: Water; Lane P: plasmid pCAMBI1301::IbNCED1 as a positive control; Lane WT: Xushu22 plant as a negative control. (G) GUS analysis of the transgenic plants. (H) qRT-PCR analysis of IbNCED1 in the transgenic plants. ** indicates a significant difference from that of WT at p < 0.01 according to Student’s t-test.

Figure 4

Plant height of transgenic sweet potato plants and WT. (A1,A2) Phenotypes and plant height of in vitro-grown transgenic sweet potato plants and WT cultured on MS medium for 4 weeks. The data are presented as the means ± SEs (n = 5). (B1,B2) Phenotypes and plant height of transgenic sweet potato plants and WT grown in transplanting boxes for 6 weeks. Bar = 10 cm. The data are presented as the means ± SEs (n = 5). (C1,C2)The histological analysis and cell length of in vitro-grown transgenic sweet potato plants and WT cultured on MS medium for 4 weeks. Bar = 100 μm. The data are presented as the means ± SEs (n = 20). The data are presented as the means ± SEs (n = 5). The different small letters indicate a significant difference at p < 0.05 according to Student’s t-test.

Figure 5

The analysis of KEGG pathway in transgenic sweet potato (A) and the expression analysis of DEGs in GA signalling pathway (B).

Figure 6

Plant height of transgenic sweet potato plants and WT after GA3 treatment. (A1–A3) Phenotypes and plant height of in vitro-grown transgenic sweet potato plants and WT cultured on MS medium for 6 weeks. (B1–B3) Phenotypes and plant height of in vitro-grown transgenic sweet potato plants and WT cultured on MS medium with 10 ng L⁻¹ GA3 for 6 weeks. Bar = 10 cm. The data are presented as the means ± SEs (n = 3). The different small letters indicate a significant difference at p < 0.05 according to Student’s t-test.