ApGA20ox1 Positively Regulates Flowering of Agapanthus praecox via the Gibberellin Metabolic Pathway

doi:10.13560/j.cnki.biotech.bull.1985.2026-0356

Abstract

Abstract:

Objective To elucidate the mechanism by which ApGA20ox1, a key gibberellin (GA) biosynthesis gene, regulates flowering in Agapanthus praecox, and to provide a theoretical basis for flowering regulation and genetic improvement in this species. Method Total RNA was extracted from leaves of A. praecox, and the full-length coding sequence of ApGA20ox1 was cloned using RACE (rapid amplification of cDNA ends). Sequence homology, subcellular localization, and prokaryotic expression were analyzed. The gene was heterologously overexpressed in Arabidopsis thaliana, whereas its endogenous expression was silenced in A. praecox via RNA interference (RNAi). Changes in GA contents and expression patterns of flowering-related genes were investigated through transcriptome sequencing and GA-targeted metabolomics. Result The ApGA20ox1 gene was successfully cloned from A. praecox. Its full-length ORF was 1 086 bp, encoding a protein of 361 amino acids, and the genomic sequence contained three exons and two introns. The gene was evolutionarily conserved, and its encoded protein was predicted to have a molecular weight of approximately 41.1 kD and to localize in the cytoplasm. Genetic transformation assays demonstrated that heterologous overexpression of ApGA20ox1 significantly advanced bolting time in A. thaliana. RNAi-mediated suppression of ApGA20ox1 expression significantly affected the expression of GA synthase genes and metabolic flux in A. praecox leaves. Among these genes, the expression levels of GA20ox6 and GA3ox1 were significantly upregulated, whereas those of GA20ox1, GA20ox3, GA20ox4, and GA2ox3 were markedly downregulated. The contents of GA₄, GA₂₄, GA₉, GA₄₄, GA₁₉, and GA₂₀ decreased significantly, whereas those of GA₃, GA₅₁, GA₂₉, and GA₃₄ increased remarkably. Among flowering-related genes, all members of the FLC and SOC1 families were significantly downregulated, whereas most members of the CO family were upregulated. Additionally, the expression of FT and LFY genes was significantly upregulated. Conclusion ApGA20ox1 was identified as a critical gene that regulates GA biosynthesis and positively regulates flowering through the GA pathway in A. praecox. RNAi-mediated silencing of ApGA20ox1 markedly repressed the expression of the flowering integrator gene SOC1 and blocked the flowering pathway.

Key words: Agapanthus praecox, ApGA20ox1, gibberellin, flowering regulation, SOC1, heterologous overexpression, targeted metabolomics, transcriptome

YUE Jian-hua, FANG Ting-ting, CHEN Guan-qun, SHEN Xiao-hui. ApGA20ox1 Positively Regulates Flowering of Agapanthus praecox via the Gibberellin Metabolic Pathway[J]. Biotechnology Bulletin, 2026, 42(11): 186-195.

Figures/Tables 6

Fig. 1 Analysis of exon-intron structure of ApGA20ox1 from A. praecoxATG and TGA are the start codon and stop codon, respectively

Fig. 2 Phylogenetic analysis of ApGA20ox1 in A. praecox

Fig. 3 Subcellular localization of ApGA20ox1 in N. benthamiana (bar 100 μm)

Fig. 4 Effect of ApGA20ox1 overexpression on flowering in A. thalianaA: PCR detection of transgenic plant DNA (M: DL2 000 DNA marker; Col-0: wild-type plant; 1-8: overexpression lines). B: Detection of ApGA20ox1 expression in wild-type and four overexpression lines of A. thaliana. C: Effect of overexpression of ApGA20ox1 on flowering in A. thaliana. D: Statistical analysis of bolting time promoted by overexpression of ApGA20ox1 in A. thaliana. * indicates significant differences compared with the control (P<0.05). The same below

Fig. 5 Effect of RNAi-mediated silencing of ApGA20ox1 on flowering in A. praecoxA: Construction of vector pTCK303-ApGA20ox1. B: PCR detection of transgenic RNAi plants (CK: wild-type plant; 1-14: RNAi lines). C: Effect of silencing of ApGA20ox1 on flowering in A. praecox. D: Detection of relative expression of ApGA20ox1

Fig. 6 Effects of silencing ApGA20ox1 expression on GAs biosynthesis in A. praecoxA: Quantification of endogenous GAs contents. * indicates significant difference at P<0.05, data shown in the figure represent RNAi vs. CK. B: Expression profiles of DEGs encoding key catalytic enzymes in the late GA biosynthesis pathway, data shown in the figure represent RNAi vs. CK. C: Schematic diagram of the late-stage GA biosynthesis pathway

References 38

[1]	Simpson MG. Diversity and classification of flowering plants: amborellales, nymphaeales, austrobaileyales, magnoliids, monocots, and ceratophyllales [M]//Plant Systematics. Amsterdam: Elsevier, 2019: 187-284.
[2]	Dong Y, Zhang YY, Zhang Y, et al. The complete chloroplast genome of Agapanthus praecox (Agapanthaceae), an ornamental and medicinal plant [J]. Mitochondrial DNA Part B, 2021, 6(12): 3329-3330.
[3]	Dong Y, Du CM, Li PL, et al. Stage-dependent callus induction in Agapanthus praecox: multi-omics reveals the semi-mature pedicel explant as the optimal choice [J]. Horticulturae, 2026, 12(1): 27.
[4]	Shen WL, Lin PP, Yu ZH, et al. Analysis of the 2OG-Fe (II) oxygenase family reveals new insights associated with flowering regulation in Saccharum spontaneum [J]. Crop Sci, 2024, 64(1): 260-277.
[5]	Wang GH, Wang JJ, Yin MR, et al. CmNLP7 interacts with CmPP6 to suppress flowering time in Chrysanthemum [J]. Hortic Plant J, 2025, 11(2): 865-876.
[6]	Zhao B, Zhai D, Wang JW. Flowering time regulation through the lens of evolution [J]. Curr Opin Plant Biol, 2025, 85: 102734.
[7]	张长生, 魏滔, 周玉萍, 等. FLC调控植物成花的分子机制研究新进展 [J]. 植物学报, 2021, 56(6): 651-663.
	Zhang CS, Wei T, Zhou YP, et al. Progress in flowering regulation mechanisms of FLC [J]. Chin Bull Bot, 2021, 56(6): 651-663.
[8]	Kang DR, Dai SL, Wang ZC. MBD protein recognizes flower control genes regulated by DNA methylation in Chrysanthemum lavandulifolium [J]. Ornam Plant Res, 2022, 2(1): 1-14.
[9]	Huang ZW, Liu GQ, Chen R, et al. The RhSPL4-RhPRR5L module positively regulates flowering time in rose (Rosa hybrida) [J]. Hortic Plant J, 2025, 11(5): 1930-1942.
[10]	牛钰凡, 陈玲玲, 苏晨, 等. 成花素FT: 环境感知与发育调控的信号枢纽 [J]. 植物学报, 2026, 61(1): 1-8.
	Niu YF, Chen LL, Su C, et al. Florigen FT: a signaling hub connecting environmental cues and developmental regulation [J]. Chin Bull Bot, 2026, 61(1): 1-8.
[11]	Izawa T. What is going on with the hormonal control of flowering in plants? [J]. Plant J, 2021, 105(2): 431-445.
[12]	Feng JY, Li JG, Zhang ZM, et al. DELLA gene expression may be involved in chasmogamous-cleistogamous flower development and regulate the expression of B-class floral homeotic genes in Viola philippica [J]. Plant Physiol Biochem, 2026, 231: 111031.
[13]	Wang M, Li AM, Pan YQ, et al. Gibberellin biosynthesis gene ScGA20 oxidase enhances sugarcane growth by modulating genes associated with phytohormone and growth processes [J]. Plant Physiol Biochem, 2025, 221: 109652.
[14]	Wang XR, Zhang YJ, Cui CY, et al. Mitochondrial NADPH dehydrogenase B1 is involved in flowering regulation through affecting gibberellin signaling pathway in Arabidopsis [J]. Plant Physiol Biochem, 2025, 229: 110814.
[15]	Barker R, Fernandez Garcia MN, Powers SJ, et al. Mapping sites of gibberellin biosynthesis in the Arabidopsis root tip [J]. New Phytol, 2021, 229(3): 1521-1534.
[16]	Qin TJ, Huang Q, Li JJ, et al. Comprehensive characterization of gibberellin oxidase gene family in Brassica napus reveals BnGA2ox15 involved in hormone signaling and response to drought stress [J]. Int J Biol Macromol, 2024, 282: 136822.
[17]	Liu XJ, Wang JY, Sun WX, et al. Sweet cherry PavGA20ox-2 positive regulation of plant growth, flowering time, and seed germination [J]. Sci Hortic, 2023, 322: 112405.
[18]	覃悦, 杨妍, 张磊, 等. 二倍体和四倍体马铃薯StGAox基因鉴定与比较分析 [J]. 生物技术通报, 2025, 41(3): 146-160.
	Qin Y, Yang Y, Zhang L, et al. Identification and comparative analysis of the StGAox genes in diploid and tetraploid potatoes [J]. Biotechnol Bull, 2025, 41(3): 146-160.
[19]	Ren M, Wang Q, Zhang FH, et al. Genome-wide identification of the GAox gene family and functional characterization of PbGA3ox4 during stone cell formation in Chinese white pear [J]. Sci Hortic, 2024, 330: 113063.
[20]	Chen Q, Yong SY, Xu F, et al. EjGASA6 promotes flowering and root elongation by enhancing gibberellin biosynthesis [J]. J Integr Agric, 2024, 23(5): 1568-1579.
[21]	Jia XQ, Yu LY, Tang ML, et al. Pleiotropic changes revealed by in situ recovery of the semi-dwarf gene sd1 in rice [J]. J Plant Physiol, 2020, 248: 153141.
[22]	Kim D, Abdelaziz ME, Ntui VO, et al. Colonization by the endophyte Piriformospora indica leads to early flowering in Arabidopsis thaliana likely by triggering gibberellin biosynthesis [J]. Biochem Biophys Res Commun, 2017, 490(4): 1162-1167.
[23]	陈香波, 张冬梅, 申瑞雪. 百子莲属品种资源综合评价与筛选 [J]. 上海农业学报, 2023, 39(4): 17-24.
	Chen XB, Zhang DM, Shen RX. Comprehensive evaluation and suitable varieties selection of Agapanthus based on AHP method [J]. Acta Agric Shanghai, 2023, 39(4): 17-24.
[24]	Li YH, Huang HL, Shi YM, et al. Genome-wide identification, evolution and expression analyses of GA2ox gene family in Brassica napus L [J]. Phyton, 2023, 92(3): 815-835.
[25]	Qi WJ, Chang JZ, Bu HH, et al. Comprehensive analysis of ZmGAox gene family in maize (Zea mays L.) reveals novel structural features and evolutionary patterns under positive selection [J]. Genet Resour Crop Evol, 2025, 72(8): 9683-9708.
[26]	Yamaguchi S. Gibberellin metabolism and its regulation [J]. Annu Rev Plant Biol, 2008, 59: 225-251.
[27]	Meng Y, Cai L, Liang DR, et al. Genome-wide identification, characterization, and expression analysis of the gibberellin-20-oxidase gene family in Artemisia annua L. [J]. Genet Resour Crop Evol, 2025, 72(4): 4149-4160.
[28]	Zhao W, Sun XX, Wu SP, et al. MaGA20ox2f, an OsSD1 homolog, regulates flowering time and fruit yield in banana [J]. Mol Breed, 2025, 45(1): 12.
[29]	Yue JH, Zhang D, Chen GQ, et al. ApGA20ox1, a key gibberellin biosynthesis gene, regulates somatic embryogenesis and plant height in Agapanthus praecox [J]. Sci Hortic, 2023, 312: 111846.
[30]	Cai XY, Wang YY, Wen YX, et al. Overexpression of pineapple gibberellin 2-oxidase gene AcGA2ox6 in Arabidopsis induces delayed flowering, dwarfism, and enhanced leaf serration [J]. Plant Physiol Biochem, 2025, 229(Pt A): 110613.
[31]	Bao SJ, Hua CM, Shen LS, et al. New insights into gibberellin signaling in regulating flowering in Arabidopsis [J]. J Integr Plant Biol, 2020, 62(1): 118-131.
[32]	Zeng XL, Lv XC, Liu R, et al. Molecular basis of CONSTANS oligomerization in FLOWERING LOCUS T activation [J]. J Integr Plant Biol, 2022, 64(3): 731-740.
[33]	Sri T, Gupta B, Tyagi S, et al. Homeologs of Brassica SOC1, a central regulator of flowering time, are differentially regulated due to partitioning of evolutionarily conserved transcription factor binding sites in promoters [J]. Mol Phylogenet Evol, 2020, 147: 106777.
[34]	Moon J, Suh SS, Lee H, et al. The SOC1 MADS-box gene integrates vernalization and gibberellin signals for flowering in Arabidopsis [J]. Plant J, 2003, 35(5): 613-623.
[35]	Chen M, Zhang TL, Zhang WB, et al. miR156-SPLs module regulates flowering and controls plant height by modulating gibberellin biosynthesis in Citrus [J]. Plant Biotechnol J, 2025, 23(10): 4271-4289.
[36]	Zhang H, Li JY, Liu WW, et al. Diversity of gibberellins contributes to different flowering ecotypes in chrysanthemum [J]. Sci Hortic, 2024, 338: 113702.
[37]	Liu P, Feng WM, Yang H, et al. Untargeted metabolomics and functional analyses reveal that the secondary metabolite quinic acid associates with Angelica sinensis flowering [J]. BMC Plant Biol, 2025, 25(1): 72.
[38]	Nasim Z, Karim N, Ahn JH. Flowering in space: transcriptional insights into the crosstalk between aging, gibberellin, and sugar pathways to modulate flowering in space [J]. Physiol Plant, 2025, 177(4): e70460.