The zinc/iron-regulated transporter-like protein (ZIP) gene family first identified in plants is highly distributed in the plant kingdom. This family has previously been reported to transport several essential and non-essential cationic elements, including those toxic to many economically important crops such as cacao (Theobroma cacao L.). In this article, we present a detailed study on physicochemical properties, evolution, duplication, gene structure, promoter region and TcZIP family three-dimensional protein structure. A total of 11 TcZIP genes have been identified to encode proteins from 309 to 435 aa, with localization in the plasma membrane and chloroplast, containing 6-9 putative domains (TM). Interspecies phylogenetic analysis subdivided the ZIP proteins into four groups. Segmental duplication events significantly contributed to the expansion of TcZIP genes. These genes underwent high pressure of purifying selection. The three-dimensional structure of the proteins showed that α helix conformations are predominant with several pocket sites, containing the metal binding site, with the residues leucine (LEU), alanine (ALA), glycine (GLY), serine (SER), lysine (LYS) and histidine (HIS) the most predicted. Regarding the analysis of the protein-protein interaction and enrichment of the gene ontology, four biological processes were assigned, the most important being the cation transport. These new discoveries expand the knowledge about the function, evolution, protein structures and interaction of ZIP family proteins in cacao and contribute to develop cacao genotypes enriched with important mineral nutrients as well as genotypes that bioaccumulate or exclude toxic metals.

Sex segregation increases the cost of Carica papaya production through seed-based propagation. Therefore, in vitro techniques are an attractive option for clonal propagation, especially of hermaphroditic plants. Here, we performed a temporal analysis of the proteome of C. papaya calli aiming to identify the key players involved in embryogenic callus formation. Mature zygotic embryos used as explants were treated with 20 μM 2,4-dichlorophenoxyacetic acid to induce embryogenic callus. Total proteins were extracted from explants at 0 (zygotic embryo) and after 7, 14, and 21 days of induction. A total of 1407 proteins were identified using a bottom-up proteomic approach. The clustering analysis revealed four distinct patterns of protein accumulation throughout callus induction. Proteins related to seed maturation and storage are abundant in the explant before induction, decreasing as callus formation progresses. Carbohydrate and amino acid metabolisms, aerobic respiration, and protein catabolic processes were enriched throughout days of callus induction. Protein kinases associated with auxin responses, such as SKP1-like proteins 1B, accumulated in response to callus induction. Additionally, regulatory proteins, including histone deacetylase (HD2C) and argonaute 1 (AGO1), were more abundant at 7 days, suggesting their role in the acquisition of embryogenic competence. Predicted protein–protein networks revealed the regulatory role of proteins 14-3-3 accumulated during callus induction and the association of proteins involved in oxidative phosphorylation and hormone response. Our findings emphasize the modulation of the proteome during embryogenic callus initiation and identify regulatory proteins that might be involved in the activation of this process.

The induction of somatic embryogenesis is controlled by various genes and proteins involved in hormonal pathways and stress responses, which act as key regulators of in vitro cellular reprogramming. In this study, we employed a temporal proteomic approach to investigate the underlying molecular mechanisms governing sugarcane (Saccharum spp.) embryogenic callus formation in response to 2,4-dichlorophenoxyacetic acid (2,4-D) during induction. Proteomic profiling revealed 996 differentially accumulated proteins (DAPs) across at least one pairwise comparison among time points (0, 7, 14 and 21 days) during callus induction. These DAPs were classified into different clusters on the basis of their accumulation profile. Proteins involved in embryogenesis, histone epigenetic regulation, hormone responses and protein post-translational modification accumulate during callus induction. The predicted interactions between the TOPLESS protein and auxin response proteins (SKP1, CUL1 and CAND1) are associated with increased accumulation of the histone deacetylase HDT2 protein, a regulator of chromatin condensation, during embryogenic callus initiation. Moreover, proteomic analysis revealed a temporal reduction in methylation cycle enzymes during callus induction, whereas global DNA methylation showed only a slight, non-significant increase, suggesting that additional regulatory layers are present. The identified protein dynamics provide valuable targets for refining somatic embryogenesis protocols and advancing their biotechnological applications in sugarcane. SIGNIFICANCE: Genetic engineering and plant cloning usually involve the induction of embryogenic competence using 2,4-dichlorophenoxyacetic acid (2,4-D). This study presents protein-protein interaction (PPI) networks regulated during the induction of sugarcane callus using 2,4-D, in addition to the morphological aspects of the explant during the process. Proteomic analysis of time series shows the regulation of protein kinases and transcriptional regulators TOPLESS, CUL1, SKP1, CAND1, and ARGONAUTE kinases, revealing mechanisms of activation of induction and multiplication of embryogenic callus. Furthermore, the possible interaction between GH3.8 and SnRK/SAPK kinases suggests a link between hormonal responses.

A embriogênese somática em cana-de-açúcar possibilita propagação de mudas em larga-escala e a regeneração de plantas geneticamente modificadas, representando uma ferramenta biotecnológica fundamental