Tomato MBD5, a methyl CpG binding domain protein, physically interacting with UV‐damaged DNA binding protein‐1, functions in multiple processes

Abstract

SummaryIn tomato (Solanum lycopersicum), high pigment mutations (hp-1 and hp-2) were mapped to genes encoding UV-damaged DNA binding protein 1 (DDB1) and de-etiolated-1 (DET1), respectively.Here we characterized a tomato methyl-CpG-binding domain protein SlMBD5 identified by yeast two-hybrid screening using SlDDB1 as a bait.Yeast two-hybrid assay demonstrated that the physical interaction of SlMBD5 with SlDDB1 is mediated by the C-termini of SlMBD5 and the b-propeller-C (BPC) of SlDDB1. Coimmunoprecipitation analyses revealed that SlMBD5 associates with SlDDB1-interacting partners including SlDET1, SlCUL4, SlRBX1a and SlRBX1b in vivo. SlMBD5 was shown to target to nucleus and dimerizes via its MBD motif. Electrophoresis mobility shift analysis suggested that the MBD of SlMBD5 specifically binds to methylated CpG dinucleotides but not to methylated CpHpG or CpHpH dinucleotides. SlMBD5 expressed in protoplast is capable of activating transcription of CG islands, whereas CUL4/DDB1 antagonizes this effect. Overexpressing SlMBD5 resulted in diverse developmental alterations including darker green fruits with increased plastid level and elevated pigmentation, as well as enhanced expression of SlGLK2, a key regulator of plastid biogenesis.Taken together, we hypothesize that the physical interaction of SlMBD5 with the CUL4-DDB1-DET1 complex component may affect its binding activity to methylated DNA and subsequently attenuate its transcription activation of downstream genes.

Like Heterochromatin Protein 1b represses fruit ripening via regulating the H3K27me3 levels in ripening‐related genes in tomato

“…The blocked membranes were incubated with anti‐HA, anti‐FLAG or anti‐H3K27me3 for another 2 h, respectively. These membranes were then washed three times for 10 min with 1× TBST buffer and incubated for 1 h with appropriate horseradish peroxidase (HRP)‐conjugated secondary antibody in blocking buffer (Li et al , 2015). Enhanced chemiluminescence detection was performed as recommended by the manufacturer (Millipore Corp.).…”

Heterologous Expression of SvMBD5 from Salix viminalis L. Promotes Flowering in Arabidopsis thaliana L.

“…MBD6 not only participates in RNA-mediated gene silencing in combination with 40S ribosomal protein AtRPS2C, AtAGO4, and nuclear transport factor AtNTF2, but it also binds to the histone deacetylase AtHDA6 in the RdDM pathway [32]. Studies in tomato (Solanum lycopersicum L.) have shown that MBD5 interacts with the CUL4-DDB1-DET1 complex, affecting its assembly on methylated DNA and thereby impairing the transcriptional activation of downstream genes [33].The different molecular functions of MBD proteins indicate that they play different roles in plant growth and development. In Arabidopsis, MBD genes are expressed in a tissue-specific manner, except for AtMBD11, which is highly expressed in all tissues [30].…”

“…Arabidopsis MBD7-1 mutants have low SUCROSE-PROTON SYMPORTER2 (SUC2) expression and develop long roots when cultivated on a sucrose-containing medium [31]. Overexpressing SlMBD5 in tomato led to dark fruit color and dwarf plants [33].To date, few studies have focused on MBD proteins in plants, especially woody plants. In the current study, we cloned the coding region of the MBD gene SvMBD5 from the woody plant Salix viminalis L. and analyzed its expression pattern and biological function.…”

“…MBD6 not only participates in RNA-mediated gene silencing in combination with 40S ribosomal protein AtRPS2C, AtAGO4, and nuclear transport factor AtNTF2, but it also binds to the histone deacetylase AtHDA6 in the RdDM pathway [32]. Studies in tomato (Solanum lycopersicum L.) have shown that MBD5 interacts with the CUL4-DDB1-DET1 complex, affecting its assembly on methylated DNA and thereby impairing the transcriptional activation of downstream genes [33].…”

“…Arabidopsis MBD7-1 mutants have low SUCROSE-PROTON SYMPORTER2 (SUC2) expression and develop long roots when cultivated on a sucrose-containing medium [31]. Overexpressing SlMBD5 in tomato led to dark fruit color and dwarf plants [33].…”