Time-Dependent Proteomic Signatures Associated with Embryogenic Callus Induction in Carica papaya L

Abstract

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.

Keywords: Carica papaya; bottom-up proteomics; embryogenic callus; micropropagation; somatic embryos; time-series analysis.

Figure 1

Morphological features of the explant during embryogenic callus induction using 2,4-D. Zygotic embryo used before incubation at time 0 (A). Initial cotlyedon folding in the induction medium after 7 days of inoculation (B), followed by callus formation after 14 days of inoculation (C), and proliferation after 21 days of inoculation (D). The white bar in the images represents 0.5 cm.

Figure 2

Results of the comparative analysis of proteomics data. Evaluation of the protein clusters revealed four patterns of accumulation throughout the time of callus induction. This analysis revealed proteins that increased in accumulation at all time points during callus induction (Cluster 1) and proteins that decreased their abundance throughout this process (Cluster 2). In addition, there are also groups of proteins that were abundant at the beginning of callus formation (7 days of induction) (Cluster 3), and proteins which accumulated after callus formation, at 21 days of incubation in the induction medium (Cluster 4).

Figure 3

Gene ontology and KEGG pathways enrichment analysis among differentially accumulated proteins. The figure shows biological processes (A), cellular components (B), molecular function (C), and KEGG pathways (D) enriched among the different protein clusters differentially accumulated during embryogenic callus induction. The analysis shows that biological energy-generating processes are regulated during embryogenic callus induction, in addition to the accumulation of proteins with regulatory functions. In addition, several metabolic pathways are activated during embryogenic callus formation, including the metabolism of amino acids and reserve consumption.

Figure 4

Protein–protein interaction network constructed with regulated DAPs during embryogenic callus induction. The network was constructed in STRING v.11.5 with C. papaya proteins. The names of the Arabidopsis orthologs are in parentheses. The color at the nodes indicates hormone response proteins (yellow), oxidative phosphorylation (blue), 14-3-3 (red), and SAM metabolism (green). The protein–protein interaction analysis highlights an important role for the 14-3-3 proteins in connecting hormonal signaling responses with energy metabolism during embryogenic callus induction. The network was constructed with medium confidence (combined score > 0.4).