Investigation of OfbHLH105 in Regulation of Carotenoid Metabolism in Osmanthus fragrans

doi:10.16420/j.issn.0513-353x.2025-0380

Acta Horticulturae Sinica ›› 2026, Vol. 53 ›› Issue (8): 2333-2344.doi: 10.16420/j.issn.0513-353x.2025-0380

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

Investigation of OfbHLH105 in Regulation of Carotenoid Metabolism in Osmanthus fragrans

WANG Yi’nan, PENG Lin, WANG Yiguang^*(), XIAO Zheng, DENG Jinping, ZHAO Hongbo^*()

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

Received:2026-04-22 Revised:2026-06-15 Online:2026-08-25 Published:2026-08-25
Contact: WANG Yiguang, ZHAO Hongbo

Abstract

Abstract:

Flower color is one of the important ornamental traits of Osmanthus fragrans. The composition and content of carotenoids in petals directly affect flower color，and their accumulation is regulated by transcription factors. In this study，the transcription factor OfbHLH105 was cloned from the O. fragrans cultivar‘Jinqiugui’. Multiple sequence alignment and phylogenetic analysis showed that OfbHLH105 contains a typical bHLH conserved domain and is most closely related to AtbHLH105 in Arabidopsis thaliana. Subcellular localization analysis indicated that OfbHLH105 is a nucleus-localized transcription factor. Expression analysis during the flowering process revealed that OfbHLH105 has the highest expression at the bud cracking stage. In transgenic tobacco plants over-expressing OfbHLH105，the carotenoid content in leaves increased，accompanied by up-regulation of carotenoid biosynthesis genes NtLCYB，and down-regulation of several downstream biosynthesis and degradation genes. Yeast two-hybrid results showed that OfbHLH105 can form homodimers. These findings suggest that OfbHLH105 may be involved in regulation of carotenoid metabolism through homodimer formation.

Key words: Osmanthus fragrans, carotenoids, transcription factor, bHLH105, flower color

WANG Yi’nan, PENG Lin, WANG Yiguang, XIAO Zheng, DENG Jinping, ZHAO Hongbo. Investigation of OfbHLH105 in Regulation of Carotenoid Metabolism in Osmanthus fragrans[J]. Acta Horticulturae Sinica, 2026, 53(8): 2333-2344.

Figures/Tables 9

Fig. 1 Different flowering stages of flower in Osmanthus fragrans‘Jinqiugui’

Table 1 Primers used in this study

引物名称 Primer name	引物序列（5′-3′） Primer sequences	目的 Purpose
OfbHLH105-F	ATGGTTTCCCCGGAAAAAACG	基因克隆 Gene cloning
OfbHLH105-R	TTATGCAACTGGTGGGCGAA	基因克隆 Gene cloning
35S::OfbHLH105-F	CCGcatatgGTTTCCCCGGAAAAAAC	植物过表达载体构建 Construction of plant overexpression vectors
35S::OfbHLH105-R	TACCggatccTTATGCAACTGGTGGGCGAAGCA
pORE-R4-F	GACGCACAATCCCACTATCC
pORE-R4-R	TTGGGACAACTCCAGTGAAA
qRT-OfbHLH105-F	ATGTCGCTGTTCCTGAAGGG	实时荧光定量PCR（qRT-PCR） Real-time quantitative PCR
qRT-OfbHLH105-R	TTCAAGGGTTGCACAGACCA
qRT-OfACT-F	CCCAAGGCAAACAGAGAAAAAAT
qRT-OfACT-R	ACCCCATCACCAGAATCAAGAA
AD-OfbHLH105-F	GGCcatatgGTTTCCCCGGAAAAAACGAATTG	酵母双杂交 Yeast two-hybrid assay
AD-OfbHLH105-R	CGGggatccTTATGCAACTGGTGGGCGAAGCA
BD-OfbHLH105-F	CCcatatgATGGTTTCCCCGGAAAAAAC
BD-OfbHLH105-R	CGggatccCTTATGCAACTGGTGGGCGAAG

Fig. 2 Cloning and bioinformatics analysis of the OfbHLH105 gene A：Gel electrophoresis of gene cloning；B：Multiple sequence alignment of OfbHLH105 amino acid sequences；C：Secondary structure prediction of the OfbHLH105 protein；D：Hydrophobicity analysis of the OfbHLH105 protein. Of：Osmanthus fragrans；Oe：Olea europaea；Nt：Nicotiana tabacum；At：Arabidopsis thaliana

Fig. 3 Phylogenetic analysis of OfbHLH105 and Arabidopsis thaliana bHLH proteins

Fig. 4 Expression levels of OfbHLH105 during the flowering process of‘Jinqiugui’ S1：Round bead stage；S2：Bud cracking stage；S3：Linggeng stage；S4：Initial flowering stage；S5：Full flowering stage；S6：Late flowering stage. Different lowercase letters indicate significant differences between flowering stages at the P < 0.05 level

Fig. 5 Subcellular localization of OfbHLH105 protein in Nicotiana benthamiana 35S::GFP is the control

Fig. 6 Phenotypic analysis and carotenoid content in leaves of tobacco overexpressing OfbHLH105 A：Phenotypic analysis of tobacco；B：RT-PCR analysis；C：Determination of total carotenoid content. ** P < 0.01

Fig. 7 Relative expression of genes in carotenoid metabolic pathway in OfbHLH105 overexpressed tobacco strains *P < 0.05，**P < 0.01.

Fig. 8 Yeast two-hybrid assay between OfbHLH105 proteins Positive control：ADT7 + BD53；Negative control：ADT7 + BDlam

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Investigation of OfbHLH105 in Regulation of Carotenoid Metabolism in Osmanthus fragrans

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