Scientia Agricultura Sinica ›› 2023, Vol. 56 ›› Issue (13): 2574-2585.doi: 10.3864/j.issn.0578-1752.2023.13.011

• HORTICULTURE • Previous Articles     Next Articles

The Function of PmABCG9 Transporter Related to the Volatilization of Benzyl Alcohol in Prunus mume

HAO RuiJie1(), QIU Chen2, GENG XiaoYun1, JIA HaoTian1, ZHANG YaJing1, CHANG Jun1, FENG XinXin1   

  1. 1 College of Horticulture, Shanxi Agricultural University, Taigu 030801, Shanxi
    2 College of Urban and Rural Construction, Shanxi Agricultural University, Taigu 030801, Shanxi
  • Received:2022-09-23 Accepted:2023-01-12 Online:2023-07-01 Published:2023-07-06
  • Contact: HAO RuiJie

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Abstract:

【Objective】The aim of this study was to improve the volatilization mechanism of floral scent by exploring the gene related to the transmembrane transport of important aromatic components in P. mume. 【Method】The P. mume Caizhiwufen was applied as material plant. Based on the analysis of volatile and endogenous contents of benzyl alcohol at different blooming stages, the ABC transporters (ATP-binding cassette transporter) involved in the transmembrane transport of benzyl alcohol were identified by weighted gene co-expression network analysis (WGCNA), phylogenetic analysis, and gene expression profiles. Finally, the functions of PmABCG9 were validated by subcellular localization, transgenic technology and incubation assay with substrates. 【Result】The flowering process of P. mume was divided into seven blooming stages, and the total relative content of benzaldehyde, benzyl alcohol and benzyl acetate reached more than 80% at the initial blooming stage, indicating that they were the key aromatic components of Caizhiwufen. The quantitative analysis showed that the endogenous content of benzaldehyde was the highest among the above three, while its volatile content was only 13.17 ng·g-1·h-1 at the highest level. The endogenous and volatile content of benzyl alcohol reached the highest at wither stage, up to 82.28 ng·g-1·h-1 of the volatile content. The highest volatile content of benzyl acetate was at full blooming stage and up to 280.21 ng·g-1·h-1. WGCNA between the transcriptome data of different blooming stages and the volatile content of aromatic components showed that the turquoise module highly correlated with the volatile content of benzyl alcohol, importantly. 11 ABCG subfamily genes were identified. Moreover, based on the expression patterns of PmABCG9 in different flower organs and blooming stages of P. mume, it was inferred that PmABCG9 was related to the transmembrane transport of benzyl alcohol in P. mume. Therefore, PmABCG9 gene was cloned, and the typical NBD-TMD domain of PmABCG9 gene was confirmed. Subcellular localization showed that PmABCG9 was localized on the cell membrane. The leaves of transgenic PmABCG9 Nicotiana benthamiana and wild-type plants were incubated with benzyl alcohol solution, and the volatile content of benzyl alcohol from each line were analyzed by GC-MS. It was noted that the volatile content of benzyl alcohol from transgenic tobaccos was significantly higher than that of wild-type ones, demonstrating that PmABCG9 had the function of transporting benzyl alcohol. 【Conclusion】 Benzaldehyde, benzyl alcohol and benzyl acetate were the key aromatic components at florescence of P. mume Caizhiwufen. PmABCG9 belonged to the semi-molecular transporters type from ABCG subfamily, PmABCG9 was closely related to volatile content benzyl alcohol, furthermore, it could effectively transport benzyl alcohol into the air through cell membrane.

Key words: Prunus mume, benzyl alcohol, PmABCG9, transport, floral scent

Fig. 1

Flower phenotypes of Caizhiwufen at different blooming stages LB: Dew petals stage; XL: Small-bud stage; ZL: Mid-bud stage; DL: Big-bud stage; CH: Initial blooming stage; SH: Full blooming stage; MH: Wither stage. The same as below"

Table 1

Primer sequence and purpose"

引物名称
Primer name		 引物序列
Prime sequence (5′-3′)		 用途
Purpose
PmABCG9-F TTGGAGAGGACACGC*ATGGAATTGCCAGTGACAATGC PmABCG9克隆、超表达
Cloning and overexpression of PmABCG9
PmABCG9-R GCAGGACTCTAGGGACTAGTCTCTGTTTCTGCAGCAT
PmABCG9-GFP-F AACACGGGGGACTTTGCAACATGGAATTGCCAGTGACAATGCCGG PmABCG9亚细胞定位
Subcellular localization of PmABCG9
PmABCG9-GFP-R CCTGAAGCGGCCGCTGTACAGTCTCTGTTTCTGCAGCATCTGTACC
PmABCG9-qPCR-F GGTTGGTTGGATTAAGGAGGAA 实时荧光定量
qRT-PCR
PmABCG9-qPCR-R CACTGAAACAAGCCACAACAG
UBC-F GCAGGTGGAGTGTTCCTTGTGA 内参引物
Internal reference primer
UBC-R GGCAGGGCTCCACTGTTCTTT

Fig. 2

The variety about content of aromatic components at different blooming stages of Caizhiwufen A: The relative content of benzaldehyde, benzyl alcohol and benzyl acetate at different blooming stages of Caizhiwufen; B: The volatile and endogenous content of benzaldehyde at different blooming stages; C: The volatile and endogenous content of benzyl alcohol at different blooming stages; D: The volatile and endogenous content of benzyl acetate at different blooming stages. Different capital and lowercase letters indicate significant difference in volatile content and endogenous content, respectively (P<0.05). The same as below"

Fig. 3

Correlation between volatiles and the genes of modules at different blooming stages of P. mume The numbers in the modules represent the value of the correlation coefficient between the module and the phenotype, and the numbers in parentheses represent the significance P value"

Fig. 4

Phylogenetic analysis of PmABCGs"

Fig. 5

The expression patterns of PmABCGs in P. mume"

Fig. 6

Conserved domain analysis and subcellular localization of PmABCG9 A: The conserved domain of PmABCG9; B: The prediction of PmABCG9 transmembrane protein structure; C: The subcellular localization of PmABCG9"

Fig. 7

The transgenic PmABCG9 tobacco lines and detection of its expression level A: Schematic of pHZM27-PmABCG9 vector; B: Seedlings of transgenic PmABCG9 tobacco; C: The expression of PmABCG9 in transgenic tobacco lines"

Fig. 8

PmABCG9 transgenic tobacco lines incubated with benzyl alcohol solution A: The volatile content of PmABCG9 transgenic tobacco lines; B: The endogenous content of PmABCG9 transgenic tobacco lines"

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