Anthocyanins are important specialized fruit metabolites and major pigments, whose abundance depends on co-regulation of activators and repressors, primarily transcription factors (TFs) of the MYB family. Herein, a KANADI-like TF PuKAN4 was characterized in pear. This TF could be transcriptionally up-regulated by the anthocyanin-related R2R3-MYBs PuMYB10/PuMYB114 and exhibited high expression within red-skinned pears. Interestingly, PuKAN4 repressed anthocyanin biosynthesis in transiently overexpressed pear fruit, and stable transformation in pear calli and tobacco plants. The PuKAN4 had a conserved EAR repression domain in C-terminal, while the repression function of PuKAN4 could be offset by a transcription activation domain VP64. The dual luciferase analysis proved that PuMYB114/PuMYB10 up-regulated expression of PuKAN4. Furthermore, the PuKAN4 could physically interact with PuMYB10/PuMYB114 and did not affect the combination of MYB10/MYB114-bHLH3, as demonstrated by Y2H, pull-down and firefly luciferase complementation. Thus, the PuKAN4 should play the role of active repressor, the formation of PuKAN4-PuMYB10/PuMYB114-PubHLH3 complex inhibited pear anthocyanin biosynthesis. Our findings unveiled an activator-and-repressor feedback loop between PuMYB114/PuMYB10 and PuKAN4, which possibly balance biosynthesis activity and prevents over-accumulation of phenylpropanoids.

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