VvLBD11/28 repress anthocyanins biosynthesis and intracellular transport to decrease their deposition in grape

Anthocyanins are plant secondary metabolites that provide fruit color and have vital nutritional value. LATERAL ORGAN BOUNDARIES DOMAIN (LBD) proteins are plant-specific transcription factors (TFs) that play a crucial role in growth and development. Some LBD TFs mediate the repressive effect of N/NO3- on anthocyanin accumulation; however, how LBD proteins regulate anthocyanin deposition is still unknown. Here, we identified two grape (Vitis vinifera) LBD TFs, VvLBD11 (homologous to AtLBD39) and VvLBD28 (homologous to AtLBD37/38), which play key roles in negatively regulating anthocyanin accumulation. In the nucleus, VvLBD11/28 act as TFs, interacting with a TOPLESS (TPL) protein VvTPL6 via the Ethylene-Responsive Element Binding Factor-associated Amphiphilic Repression (EAR) motif to inactivate the expression of anthocyanin structural genes such as VvUFGT and VvLDOX, and ultimately reducing anthocyanin synthesis. Additionally, the identified interacting proteins also included members of the MBW complex, which consists of MYB, bHLH (MYC), and WDR proteins, such as VvMYBA1, VvMYBA2r, and VvMYC1. These interactions disrupt the association between VvMYBA1/A2r and VvMYC1, resulting in the synthesis of the quaternary protein complex MYBA1/A2r-LBD11/28-MYC1-WDR1/2, which significantly reduced the MBW complex ability to activate the promoters of the anthocyanin structural genes. Concurrently, in the cytoplasm, VvLBD11/28 further interact with the anthocyanin transporter VvGST4, competitively binding with the same amino acid residues of anthocyanins binding sites in VvGST4, inhibiting its ability to bind and transport anthocyanins to the vacuole. Taken together, these findings elucidate a dual regulatory mechanism through which VvLBD11/28 synchronously repress anthocyanin biosynthesis and intracellular transport in grape.