Ectopic Overexpression of OfNCED3 Regulates the Synthesis of Carotenoids and Chlorophylls in Transgenic Tobacco Leaves

doi:10.16420/j.issn.0513-353x.2022-0330

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (6): 1284-1294.doi: 10.16420/j.issn.0513-353x.2022-0330

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Ectopic Overexpression of OfNCED3 Regulates the Synthesis of Carotenoids and Chlorophylls in Transgenic Tobacco Leaves

DU Yanxia, WANG Yiguang, XIAO Zheng, DONG Bin, FANG Qiu, ZHONG Shiwei, YANG Liyuan, ZHAO Hongbo()

Key Laboratory of National Forestry and Grassland Administration on Germplasm Innovation and Utilization for Southern Garden Plants，Zhejiang Provincial Key Laboratory of Germplasm Innovation and Utilization for Garden Plants，School of Landscape Architecture，Zhejiang A & F University，Hangzhou 311300，China

Received:2022-07-13 Revised:2023-02-06 Online:2023-06-25 Published:2023-06-27
Contact: * （E-mail：zhaohb@zafu.edu.cn）

Abstract

Abstract:

In this study，the OfNCED3 cDNA sequence were acquired based on the transcriptional data of Osmanthus fragrans‘Yanhong Gui’. The multiple sequence alignment analysis showed that OfNCED3 had two conservative domain（MIAHPKxDP and HDFAITE）characteristics of the NCED family and four conserved histidine residues. Phylogenetic analysis indicated that OfNCED3 is grouped together with SlNCED1. The results of real-time quantitative PCR showed that the expression of OfNCED3 in the petals of‘Yanhong Gui’with higher carotenoid content was significantly lower than that of‘Yu Linglong’with lower carotenoid content during flower opening process. It is speculated that OfNCED3 may be related to the difference of carotenoid contents between‘Yanhong Gui’and‘Yu Linglong’petals. Transgenic tobacco lines overexpressing OfNCED3 were obtained by an Agrobacterium-mediated method. The contents of chlorophyll，carotenoid and ABA were determined by using spectrophotometer and ultra high performance liquid chromatography（UPLC），and the expression levels of genes related to carotenoid pathway were analyzed via real-time quantitative PCR. The overexpression of OfNCED3 gene expression resulted in a significant decrease of chlorophyll and carotenoid contents and a increase of endogenous ABA content. The expression of carotenoid synthesis genes was significantly up-regulated，and the downstream synthesis genes were more up-regulated than the upstream synthesis genes. This research suggests OfNCED3 can promote the synthesis of ABA by degrading carotenoids in plants.

Key words: Osmanthus fragrans, NCED, carotenoids, chlorophyll, ABA

CLC Number:

S685.13

DU Yanxia, WANG Yiguang, XIAO Zheng, DONG Bin, FANG Qiu, ZHONG Shiwei, YANG Liyuan, ZHAO Hongbo. Ectopic Overexpression of OfNCED3 Regulates the Synthesis of Carotenoids and Chlorophylls in Transgenic Tobacco Leaves[J]. Acta Horticulturae Sinica, 2023, 50(6): 1284-1294.

Figures/Tables 8

Table 1 Specific primers for real-time quantitative PCR

基因 Gene	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
OfNCED3	TTCAAAGCCGCAATGGAATTTTATT	GATAGATTCTGGGATTTTCCCTGTC
OfACT	CCCAAGGCAAACAGAGAAAAAAT	ACCCCATCACCAGAATCAAGAA
Nt26S-RNA	GAAGAAGGTCCCAAGGGTTC	TCTCCCTTTAACACCAACGG

Fig. 1 Multiple alignment of amino acid sequences of OfNCED3 and NCEDs in other plants Of：Osmanthus fragrans；Sl：Solanum lycopersicum；At：Arabidopsis thaliana；Nt：Nicotiana tabacum；Cs：Citrus sinensis. The NCED motifs of MEAHPKXDP and HDFAITK are boxed. Four conserved histidines required for activity are marked by ▼.

Fig. 2 Phylogenetic tree of NCED proteins in Osmanthus fragrance and other plants

Fig. 3 Expression level of OfNCED3 gene in flower opening process of Osmanthus fragrans‘Yanhong Gui’and‘Yu Linglong’ S1：Bead stage；S2：Apical shell stage；S3：Boll stem stage；S4：Initial flowering stage. ** indicate statistically significant difference (P < 0.01); *** indicate extremely significant difference (P < 0.001).

Fig. 4 Overexpression vector (A), transformed tobacco phenotype (B) and expression level (C) of OfNCED3 in transgenic tobacco plants

Fig. 5 Effects of overexpression of OfNCED3 gene on chlorophyll（A）, carotenoid（B）and ABA（C）content in tobacco leaves Different lowercase letters indicate significant differences（P < 0.05）. The same below.

Fig. 6 Relative expression levels of carotenoid biosynthetic genes in tobacco leaves of the control and transgenic lines

Table 2 Up-regulation of relative expression levels of carotenoid biosynthetic genes in leaves of the control and transgenic lines

基因 gene	WT	OE-1	OE-2	基因 gene	WT	OE-1	OE-2	基因 gene	WT	OE-1	OE-2
NtPSY	1	2.12	2.70	NtLCYB2	1	2.55	1.41	NtCCD4-b	1	15.21	5.65
NtPDS	1	4.06	3.51	NtVDE	1	1.66	2.04	NtCCD4-c	1	5.83	5.98
NtZDS	1	4.20	3.27	NtNXC	1	0.92	2.42	NtNCED3-a	1	3.91	11.29
NtCRTISO	1	5.00	5.55	NtCCD1-a	1	0.65	7.16	NtNCED3-b	1	26.54	21.82
NtLCYE	1	5.79	3.31	NtCCD1-b	1	1.17	0.52	NtNCED5	1	3.03	2.16
NtLCYB1	1	1.02	2.02	NtCCD4-a	1	2.49	4.95

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Ectopic Overexpression of OfNCED3 Regulates the Synthesis of Carotenoids and Chlorophylls in Transgenic Tobacco Leaves

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