Identification of Litsea cubebaTCP Genes and Their Roles in the Regulation of Terpenoid Biosynthesis

doi:10.13560/j.cnki.biotech.bull.1985.2025-0740

Abstract

Abstract:

Objective To screen and validate TCP transcription factors potentially involved in regulating terpenoid biosynthesis in Litsea cubeba. Method Based on the whole-genome sequence of L. cubeba and transcriptome data from fruits at different developmental stages, bioinformatics approaches were employed to systematically analyze the physicochemical properties, chromosomal distribution, gene structures, conserved motifs, cis-acting elements, and collinearity of TCP transcription factors. RT-qPCR was applied to examine the expression patterns of CIN subfamily members in different tissues and fruit developmental stages. Subcellular localization of key transcription factors was performed, and their functions were further validated through yeast one-hybrid and transient expression assays. Result A total of 30 TCP gene family members (LcTCPs) were identified, unevenly distributed across nine chromosomes and designated LcTCP1 to LcTCP30. Their open reading frames ranged from 504 to 1 755 bp, with predicted molecular weights of 17 711.98–63 421.45 Da. These genes were classified into three subfamilies via phylogenetic analysis: PCF (17 members), CIN (7 members), and CYC/TB1 (6 members), consistent with the conserved motif patterns. Gene family expansion was mainly driven by whole-genome duplication events, and members within each subfamily had high conservation. RT-qPCR analysis revealed that LcTCP6 and LcTCP11 of the CIN subfamily showed relatively high expression in the fruits and during the peak stage of essential oil biosynthesis, suggesting their potential involvement in terpenoid metabolism. Notably, LcTCP11 showed an expression pattern similar to that of the key terpenoid biosynthetic gene LcDXS5 during fruit development and was localized in the nucleus. Functional assays confirmed that LcTCP11 regulated the expression of LcDXS5. Conclusion Thirty TCP transcription factors were identified from the L. cubeba genome. Among them, LcTCP11 showed co-expression with the key terpenoid biosynthetic gene LcDXS5 and was highly expressed in fruits compared with other tissues, suggesting that LcTCP11 may participate in regulating terpenoid biosynthesis in L. cubeba through regulating LcDXS5.

Key words: Litsea cubeba, TCP transcription factors, LcTCP11, LcDXS5, terpenoid biosynthesis, transcriptional regulation, expression pattern

NI Fei-fei, CHEN Yi-cun, GAO Ming, ZHANG Sheng-jiao, PENG Fang-you, CHEN Tao-mei, ZHAO Yun-xiao, WANG Yang-dong. Identification of Litsea cubebaTCP Genes and Their Roles in the Regulation of Terpenoid Biosynthesis[J]. Biotechnology Bulletin, 2026, 42(4): 227-238.

Figures/Tables 9

Fig. 1 Chromosomal localization of LcTCP genesBlue indicates the PCF subfamily, yellow indicates the CYC/TB1 subfamily, and red indicates the CIN subfamily

Fig. 2 Phylogenetic tree of TCP proteins from L. cubeba and A. thaliana

Fig. 3 Conserved motif and gene structure prediction of the LcTCPs proteins

Fig. 4 Synteny relationship of LcTCP genesPink boxes indicate chromosomes, blue lines indicate syntenic relationships among PCF subfamily genes, red lines indicate those of the CIN subfamily, and yellow lines indicate those of the CYC/TB1 subfamily. The gray background indicates segmentally duplicated genes across the L. cubeba genome

Fig. 5 Cis-acting element analysis of LcTCP transcription factor promoters

Fig. 6 Expression patterns in different tissues and correlation of expression at different fruit developmental stagesA: Expression patterns of 6 CIN subfamily members from L. cubeba at different fruit developmental stages and in various tissues. B: Expression correlation among LcTCP6, LcTCP11, LcTCP19, LcTCP30, and LcDXS5 at different fruit developmental stages. F1: 30 d after flowering; F2: 60 d after flowering, the peak period of essential oil synthesis; F3: 90 d after flowering; F4: 120 d after flowering. Different lower letters indicate significant differences (P<0.05)

Fig. 7 Expression analysis of LcTCP11 and LcDXS5 in transiently transformed L. cubeba leavesOE1, OE2, and OE3 refer to three overexpression lines. Data are expressed as mean±SD from three replicates (*P<0.05, **P<0.01, ***P<0.001,and ****P<0.000 1)

Fig. 8 Subcellular localization of LcTCP11A: Schematic diagram of the vector. B: Subcellular localization of the empty vector. C: Localization of LcTCP11-GFP fusion expression

Fig. 9 Y1H assays verifying the recognition of LcTCP11 to the TCP element in LcDXS5 promoterThe red text in the figure indicates the binding element sequences on the promoter and the corresponding mutated element sequences, respectively. Yeast cells co-expressing pGADT7-LcTCP11 and pAbAi-3×gtgggtcc were cultured on selective medium containing AbA (SD/-Trp/-Ura) at 30 ℃ for 48 h. Once single colonies had grown, they were diluted in water to 10¹-10⁴ fold, and 10 μL of each dilution was spotted onto fresh selective medium containing AbA (SD/-Trp/-Ura) for a further 48 h of cultivation

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