Identification Of Interacting-Proteins Of MaDREB2 Transcription Factor Related To Banana Fruit Ripening

Banana is one of the most important tropical and subtropical fruits with significant economic benefit in national and international markets. However, bananasare a typicalclimacteric fruit where once the ripening is triggered by ethylene treatment they exhibit a very short shelf life. Therefore, understanding the molecular mechanism of banana ripening is of great significance to extend the shelf life and maintain the quality of the fruit. The dehydration-responsive element-binding(DREB) proteinsare a kind of plant specific transcription factors and play an important role in the response of plant toabiotic stresses such as low temperature, drought and salt. Our previous works indicated that Ma DREBs may be involved inthe ripening of banana,among which Ma DREB2 is more closely related to this process. Ma DREB2 was found to bind to and activate the promoters of genes involved in armoa production such as Ma ADH1 and Ma PDC. Mitogen-activated protein kinase(MAPK) proteins are a kind of serine/threonine kinasesthat phosphorylate the substrates(such as enzymases and transcription factors), thus contributing to modulating plant growth and development, hormone response, biotic and abiotic stresses. In order to clarify the regulatory mechanism of banana fruit ripening mediated by Ma DREB2,the potential interactions of Ma DREB2 with Ma MAPKs as well as whether their interactions could affect Ma DREB2’s DNA-binding activitieswere investigated. Moreover, the possible interactions of Ma DREB2 with Ma E3 s, another kind of proteins involved in posttranslational modifications, were also analyzed. The main results are as follows:1.Ma MPK1-3are direct targets of Ma DREB2, which are inducd by fruit ripening and ethylene treatment.Ch IP–q PCR assays demonstrated that Ma DREB2 was capable of binding to the promoters of three Ma MPKs,Ma MPK1–3. Phylogenetic analysis showed that Ma MPK1, Ma MPK2 and Ma MPK3 belonged to two different subfamilies, in which Ma MPK1/2 were fell into subfamily D whereas MaMPK3 was grouped in subfamily A. Amino acids alignment clearly illustrated the presence of 11 conserved domains and the activation loop domain TXY in Ma MPK1-3 proteins. RT-q PCR analysis reavealed that the Ma MPK1-3genes displayed similar expression patterns in pulp of banana fruit withthree different ripening behaviors, and they were all up-regulated by fruit ripening and ethylene treatment.2. Ma MPK2 physically interacted with Ma DREB2, and the interaction attenuated Ma DREB2-mediated transcriptional activation activities.Yeast two-hybrid(Y2H) and bimolecular fluorescence complementation(Bi FC) assaysrevealed that Ma MPK2 physically interacted with Ma DREB2, and this interaction occured in the nucleus of the cells. Furthermore, transient expression assays showed that coexpression of Ma MPK2 and Ma DREB2 significantly reduced the activities of LUC reporter driven by Ma ADH1 and Ma PDC promoters, indicating that Ma MPK2 interacts with Ma DREB2 to attenuate Ma DREB2-mediated transcriptional activation activities.3. Ma E3 s could interact with Ma DREB2, whose transcripts were all increased during fruit ripening and by ethylene treatment.Three unique full-lengths of Ma E3 s, designated as Ma E3-1/2/3, were isolated from banana fruit. Alignments of the full-length deduced proteins of Ma E3-1/2/3 showed that they all belonged to RING-finger family of proteins. Y2 H and Bi FC assaysrevealed that Ma E3-2 and Ma E3-3 were able to interact with Ma DREB2. RT-q PCR analysis reavealed that the Ma E3-2 and Ma E3-3 genes displayed similar expression patterns during banana fruit ripening, with higher levels in the ripening stage.Collectivley, based on our findings, we speculated that transcription factor Ma DREB2 might be phosphorylated by Ma MPK2, and the phosphorylated form of Ma DREB2 could be recognized by Ma E3 s and was degraded through theubiquitin-proteasome system, thus reducing the activation activities of downstream target genes Ma ADH1 and Ma PDC by Ma DREB2. It will be very interesting to study the phosphorylation and ubiquitination of Ma DREB2 protein in the future.