MaMYB4 is a negative regulator and a substrate of RING-type E3 ligases MaBRG2/3 in controlling banana fruit ripening.

Fruit ripening is a complex developmental process, which is modulated by both transcriptional and post-translational events. Control of fruit ripening is important in maintaining moderate quality traits and minimizing postharvest deterioration. In this study, we discovered that the transcription factor MaMYB4 acts as a negative regulator of fruit ripening in banana. The protein levels of MaMYB4 decreased gradually with banana fruit ripening, paralleling ethylene production, and decline in firmness. DNA affinity purification sequencing combined with RNA-sequencing analyses showed that MaMYB4 preferentially binds to the promoters of various ripening-associated genes including ethylene biosynthetic and cell wall modifying genes. Furthermore, ectopic expression of MaMYB4 in tomato delayed tomato fruit ripening, which was accompanied by downregulation of ethylene biosynthetic and cell wall modifying genes. Importantly, two RING finger E3 ligases MaBRG2/3, whose protein accumulation increased progressively with fruit ripening, were found to interact with and ubiquitinate MaMYB4, contributing to decreased accumulation of MaMYB4 during fruit ripening. Transient overexpression of MaMYB4 and MaBRG2/3 in banana fruit ripening delayed or promoted fruit ripening by inhibiting or stimulating ethylene biosynthesis, respectively. Taken together, we demonstrate that MaMYB4 negatively modulates banana fruit ripening, and that MaMYB4 abundance could be regulated by protein ubiquitination, thus providing insights into the role of MaMYB4 in controlling fruit ripening at both transcriptional and post-translational levels.

Phosphorylation of transcription factor bZIP21 by MAP kinase MPK6-3 enhances banana fruit ripening.

Ripening of fleshy fruits involves both diverse post-translational modifications (PTMs) and dynamic transcriptional reprogramming, but the interconnection between PTMs, such as protein phosphorylation and transcriptional regulation, in fruit ripening remains to be deciphered. Here, we conducted a phosphoproteomic analysis during banana (Musa acuminata) ripening and identified 63 unique phosphopeptides corresponding to 49 proteins. Among them, a Musa acuminata basic leucine zipper transcription factor21 (MabZIP21) displayed elevated phosphorylation level in the ripening stage. MabZIP21 transcript and phosphorylation abundance increased during banana ripening. Genome-wide MabZIP21 DNA binding assays revealed MabZIP21-regulated functional genes contributing to banana ripening, and electrophoretic mobility shift assay, chromatin immunoprecipitation coupled with quantitative polymerase chain reaction, and dual-luciferase reporter analyses demonstrated that MabZIP21 stimulates the transcription of a subset of ripening-related genes via directly binding to their promoters. Moreover, MabZIP21 can be phosphorylated by MaMPK6-3, which plays a role in banana ripening, and T318 and S436 are important phosphorylation sites. Protein phosphorylation enhanced MabZIP21-mediated transcriptional activation ability, and transient overexpression of the phosphomimetic form of MabZIP21 accelerated banana fruit ripening. Additionally, MabZIP21 enlarges its role in transcriptional regulation by activating the transcription of both MaMPK6-3 and itself. Taken together, this study reveals an important machinery of protein phosphorylation in banana fruit ripening in which MabZIP21 is a component of the complex phosphorylation pathway linking the upstream signal mediated by MaMPK6-3 with transcriptional controlling of a subset of ripening-associated genes.

The transcriptional regulatory network mediated by banana (Musa acuminata) dehydration-responsive element binding (MaDREB) transcription factors in fruit ripening.

Fruit ripening is a complex, genetically programmed process involving the action of critical transcription factors (TFs). Despite the established significance of dehydration-responsive element binding (DREB) TFs in plant abiotic stress responses, the involvement of DREBs in fruit ripening is yet to be determined. Here, we identified four genes encoding ripening-regulated DREB TFs in banana (Musa acuminata), MaDREB1, MaDREB2, MaDREB3, and MaDREB4, and demonstrated that they play regulatory roles in fruit ripening. We showed that MaDREB1-MaDREB4 are nucleus-localized, induced by ethylene and encompass transcriptional activation activities. We performed a genome-wide chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq) experiment for MaDREB2 and identified 697 genomic regions as potential targets of MaDREB2. MaDREB2 binds to hundreds of loci with diverse functions and its binding sites are distributed in the promoter regions proximal to the transcriptional start site (TSS). Most of the MaDREB2-binding targets contain the conserved (A/G)CC(G/C)AC motif and MaDREB2 appears to directly regulate the expression of a number of genes involved in fruit ripening. In combination with transcriptome profiling (RNA sequencing) data, our results indicate that MaDREB2 may serve as both transcriptional activator and repressor during banana fruit ripening. In conclusion, our study suggests a hierarchical regulatory model of fruit ripening in banana and that the MaDREB TFs may act as transcriptional regulators in the regulatory network.

Banana Transcription Factor MaERF11 Recruits Histone Deacetylase MaHDA1 and Represses the Expression of MaACO1 and Expansins during Fruit Ripening.

Phytohormone ethylene controls diverse developmental and physiological processes such as fruit ripening via modulation of ethylene signaling pathway. Our previous study identified that ETHYLENE RESPONSE FACTOR11 (MaERF11), a transcription factor in the ethylene signaling pathway, negatively regulates the ripening of banana, but the mechanism for the MaERF11-mediated transcriptional regulation remains largely unknown. Here we showed that MaERF11 has intrinsic transcriptional repression activity in planta. Electrophoretic mobility shift assay and chromatin immunoprecipitation analyses demonstrated that MaERF11 binds to promoters of three ripening-related Expansin genes, MaEXP2, MaEXP7 and MaEXP8, as well as an ethylene biosynthetic gene MaACO1, via the GCC-box motif. Furthermore, expression patterns of MaACO1, MaEXP2, MaEXP7, and MaEXP8 genes are correlated with the changes of histone H3 and H4 acetylation level during fruit ripening. Moreover, we found that MaERF11 physically interacts with a histone deacetylase, MaHDA1, which has histone deacetylase activity, and the interaction significantly strengthens the MaERF11-mediated transcriptional repression of MaACO1 and Expansins Taken together, these findings suggest that MaERF11 may recruit MaHDA1 to its target genes and repress their expression via histone deacetylation.