CsTT8 regulates anthocyanin accumulation in blood orange through alternative splicing transcription

Abstract

A homologous gene of basic-helix–loop–helix AtTT8 in Arabidopsis thaliana was identified in juice sac cells of pulp tissues from blood orange (Citrus sinensis cv ‘Tarocco’), which was designated as CsTT8 in this study. Additionally, the mRNA levels of TT8 with the full-length open reading frame were significantly higher in ‘Tarocco’ than in mutant fruit lacking pigment in pulp or peel tissues. However, an alternative splicing transcript, Δ15- TT8, with the fourth exon skipped, was also identified from transcripts different in length from that in ‘Tarocco’. The mRNA levels of Δ15- TT8 were higher in mutant fruit lacking pigment in pulp or peel tissues than in the wild type. Therefore, the TT8/Δ15- TT8 mRNA level ratio was found to be crucial for sufficient pigment in either pulp or peel tissues. TT8 from blood orange fruit demonstrated the capacity for nucleus localization and binding to other proteins. In contrast, Δ15-TT8, lacking the fourth exon, lost its ability to interact with RUBY1 and to localize at the nucleus. Using a dual luciferase reporter assay and transient overexpression in tobacco, we proved that two regulatory complexes formed by a functional TT8 with different MYB(v-myb avian myeloblastosis viral oncogene homolog)-type partners significantly promoted expression of an anthocyanin biosynthetic gene and a proton pumping gene, leading to anthocyanin and citrate production. Our findings suggest that TT8, rather than dysfunctional Δ15-TT8, is possibly involved in modulating anthocyanin biosynthesis and its transport into vacuoles by proton gradients. However, increased mRNA levels of the dysfunctional alternative splicing transcript may act as a negative feedback to downregulate TT8 expression and limit anthocyanin accumulation in blood oranges.

Acknowledgements

This work was supported by Sichuan Province International Science and Technology Innovation and Cooperation Program (22GJHZ0183) and the Chengdu Municipality Technology Innovation and Development Program (2022-YF05-00450-SN).

Author contributions

W.J.H. and X.R. were responsible for identification of alternative splicing and transcripts. X.R., Q.S.P., W.W.C., and Z.F. carried out qPCR, yeast hybrid, subcellular localization, transient overexpression, and fruit quality experiments. W.J.H. conceived the experimental design and drafted the manuscript. W.J.H. proposed and supervised the research. All authors read and approved the final manuscript.

Data availability statement

Three transcription factors, WD40 (GenBank accession number KT757349), RUBY1 (KT757348), and TT8 (KT757350), from blood orange ‘Tarocco’ have been deposited in GenBank. The sequences of a total of 194 bHLH with conserved motif of DNA–binding superfamily proteins selected from ‘Valencia’ at the ICGC database in Peking have been deposited in the University Transcription Factor Database (http://planttfdb.gao-lab.org/index.php).

Conflict of interest

The authors declare that they have no conflict of interest.

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