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Journal of Integrative Agriculture
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Up-regulation of PsbHLH3 assists the induction of anthocyanin biosynthesis in the amber-flesh of black plums trigged by cold storage

Yubei Wang1, Ranran Xu2, Yanyan Ma1, Zhilei Zhao3, Yuhong Gu4#, Jiankang Cao1#

1 College of Food Science and Nutritional Engineering, China Agricultural University, 17 Qinghuadonglu Road, Beijing 100083, China

2 Institute of Agri-Food Processing and Nutrition, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100093, China

3 College of Quality and Technical Supervision, Hebei University, Baoding 071002, China 

4 College of Life Sciences, Hebei Agricultural University, 2596 Lekai South of Street, Baoding 071000, China

 Highlights 

Low temperature storage induced flesh reddening of the amber-fleshed plum cultivar.

10 candidate PsbHLHs differentially expressed in reddened flesh were screened out.

PsbHLH3 was validated to be crucial in cold-induced plum anthocyanin biosynthesis.

PsbHLH3 interacted with PsMYB10.1 to enhance activation on the promoter of PsUFGT.

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摘要  

黑皮琥珀色李果肉冷藏会诱导果肉中花色苷的合成与积累,引起低温后果肉的颜色变化。bHLH转录因子在响应冷诱导和花色苷生物合成中具有潜在功能。为探究bHLH基因的作用,本研究选取两个琥珀色果肉、黑色果皮的李品种(Prunus salicina Lindl. cv.‘Friar’和‘Angeleno’)作为果实材料,进行转录组学和生物信息学分析。通过 RNA 测序鉴定差异表达的bHLH基因,并利用qRT-PCR验证。在冷诱导果肉变红过程中,鉴定出10个表达变化最显著的bHLH 基因,包括5个上调和5个下调基因。对这10个bHLH蛋白的结构进行了鉴定,并构建了系统发育树。其中,evm.TU.Chr8.2504基因被鉴定为PsbHLH3,含有低温响应因子和MYB 结合位点。通过瞬时过表达实验进一步验证了PsbHLH3促进花色苷合成的功能。酵母双杂交和双分子荧光互补实验表明,PsbHLH3与PsMYB10.1互作,双荧光素酶试验证实了PsbHLH3通过促进 PsMYB10.1PsUFGT启动子的激活,进而增强花色苷合成相关结构基因的转录。本研究表明,低温诱导PsbHLH3上调表达,推动李果实果肉变红的分子开关 “开启”,阐明了PsbHLH3在采后冷藏诱导李果肉花色苷合成调控中的作用。



Abstract  

Cold storage of amber-fleshed black plums induced the biosynthesis and accumulation of anthocyanin in plum fruit, which caused the color change of the flesh tissue when stored at low temperature. bHLH transcription factors had potential functions in response to cold induction and anthocyanin biosynthesis. To investigate the role of bHLH genes, two amber-fleshed and black-skinned cultivars (Prunus salicina Lindl. cv. Friar and cv. Angeleno) were selected as fruit material to conduct transcriptomic and bioinformatics analysis. The differentially expressed bHLHs genes were identified via RNA sequencing and verified by qRT-PCR. Ten bHLHs genes, containing 5 up-regulated and 5 down-regulated, were identified as the most significantly altered during cold-induced flesh reddening. The structure of these 10 bHLH proteins was characterized, and a phylogenetic tree was constructed. Among them, the gene evm.TU.Chr8.2504, identified as PsbHLH3, exhibits low temperature responsiveness and contains MYB-binding sites. Further experiments involving transient overexpression were performed to validate the function of PsbHLH3 in facilitating anthocyanin biosynthesis. The yeast two-hybrid and bimolecular fluorescence complementation demonstrated that PsbHLH3 interacts with PsMYB10.1. Dual-luciferase assay showed that PsbHLH3 promoted the activation of PsMYB10.1 on the promoter of PsUFGT, and consequently augmenting the transcription of structural genes related to anthocyanin biosynthesis. The study indicated that low temperatures induced the up-regulation of PsbHLH3, facilitating the opening of ON/OFF molecular switch responsible for the flesh-reddening of plum fruit. This elucidates the role of PsbHLH3 in postharvest regulation of anthocyanin biosynthesis in plums trigged by cold storage.

Keywords:  Plum       bHLH              Flesh reddening              Anthocyanin biosynthesis              Cold storage  
Online: 08 December 2025  
Fund: 

This work was supported by the Beijing Municipal Natural Science Foundation (No. 6254032), the National Natural Science Foundation of China (No. 31872907) and S & T Program of Hebei (No. C2021201011).

Cite this article: 

Yubei Wang, Ranran Xu, Yanyan Ma, Zhilei Zhao, Yuhong Gu, Jiankang Cao. 2025. Up-regulation of PsbHLH3 assists the induction of anthocyanin biosynthesis in the amber-flesh of black plums trigged by cold storage. Journal of Integrative Agriculture, Doi:10.1016/j.jia.2025.12.009

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