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Early regulatory networks driving somatic embryogenesis in Saccharum spp. L. revealed by time-resolved proteomics

ABSTRACT: Sugarcane (Saccharum spp. hybrid) is an economically important crop that is widely grown for its sucrose content, and somatic embryogenesis is a promising approach for efficient micropropagation and genetic transformation in this context. 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 embryogenic callus formation in response to 2,4-dichlorophenoxyacetic acid (2,4-D) during induction. Comprehensive 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. Our study advances the understanding of the molecular mechanisms of somatic embryogenesis in sugarcane, highlighting the regulation of key proteins during this process. Proteins involved in photosynthesis and starch accumulation were not detected during callus induction. In contrast, 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 proteins) 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 (GDM) showed only a slight, non-significant increase, suggesting that additional regulatory layers are involved in embryogenic competence. The identified protein dynamics provide valuable targets for refining somatic embryogenesis protocols and advancing their biotechnological applications in sugarcane.

INSTRUMENT(S):

ORGANISM(S): Saccharum Hybrid Cultivar Sp80-3280

TISSUE(S): Plant Cell, Cell Culture

SUBMITTER: Vanildo Silveira

LAB HEAD: Vanildo Silveira

PROVIDER: PXD068502 | Pride | 2026-01-15

REPOSITORIES: Pride

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			Action	DRS
	2023_07_13_Inducao_cana_1.raw.zip	Raw
	2023_07_13_Inducao_cana_10.raw.zip	Raw
	2023_07_13_Inducao_cana_11.raw.zip	Raw
	2023_07_13_Inducao_cana_12.raw.zip	Raw
	2023_07_13_Inducao_cana_2.raw.zip	Raw

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Project description:Sugarcane is a crop widely used worldwide for bioenergy production, and its genetic breeding is highly dependent on tissue culture techniques. Auxin biosynthesis and transport are critical processes for in vitro tissue culture development, but the regulatory responses associated with these processes are still poorly understood in plants. We investigated the role of auxin transport during somatic embryo differentiation in sugarcane callus using an auxin transport inhibitor through a high-throughput proteomic approach. Four concentrations of 2,3,5-triiodobenzoic acid (TIBA 0, 1, 10, and 100 µM) were tested on sugarcane embryogenic callus during the pre-maturation phase of somatic embryogenesis. Pre-maturation treatment with 100 µM TIBA inhibited the differentiation of embryogenic callus into somatic embryos. Then, embryogenic callus from control and 100 µM TIBA treatments were compared via proteomic analysis. A total of 1221 proteins were found, of which 740 were differentially accumulated (DAP) in the 100 µM TIBA/Control comparison. Among the DAPs, 33 were unique to the control treatment, 31 were unique to the 100 µM TIBA pre-maturation treatment, and 415 down-regulated and 261 up-regulated. Inhibition of auxin transport impaired the accumulation and predicted protein‒protein interactions involved in embryonic development, translation, ubiquitination, and energy metabolism. The levels of spermidine synthase and spermidine were reduced, suggesting an association between auxin and polyamines during embryonic development in vitro. The down-regulation of the proteins chaperone BOBBER1, Cullin-associated NEDD8-dissociated protein 1, and Receptor-like kinase TMK2 could be associated with auxin responses and the predicted interactions between proteasome members found during the differentiation of somatic embryos. These new findings demonstrate the role of auxin transport in downstream responses during somatic embryo differentiation and open new avenues for exploring the potential of sugarcane for biotechnological purposes.

Project description:The cost of Carica papaya production through seed-based propagation is increased by sex segregation, making in vitro techniques a more appealing option for clonal propagation. Inducing embryogenic callus with 2,4-dichlorophenoxyacetic acid (2,4-D) hold the potential to large-scale cloning, although the molecular mechanisms underlying this process are still not well understood. In this study, we performed a temporal analysis in the proteome of C. papaya callus to identify the key players involved in embryogenic differentiation. Mature zygotic embryos were used as explants and treated with 20 μM 2,4-D to induce embryogenic callus. Total proteins were extracted at 0, 7, 14, and 21 days (T0, T7, T14, and T21), and 1407 proteins were identified using bottom-up proteomic approach. Comparative proteomics revealed 957 differentially accumulated proteins (DAPs) (p<0.05 and log2FC >0.585 or <-0.585) in at least one comparison between the analyzed induction times points. The clustering analysis revealed four clusters with distinct patterns of protein accumulation throughout the embryogenic callus induction treatment. The cluster 1 contains 386 DAPs that accumulated at all analyzed times after treatment with 2,4-D. In contrast, cluster 2 contains 165 DAPs that decrease in abundance during the induction. The cluster 3 contains 251 proteins that are most abundant just after the start of incubation in 2,4-D (T7) and cluster 4 grouped 155 proteins that accumulate after callus formation. Functional analysis revealed that proteins involved with reserve storage and seed maturation were more abundant in the explant at T0 and decreased as callus formation progressed. Biological processes involving carbohydrate and amino acid metabolism, aerobic respiration, and protein catabolic processes were enriched after induction treatment. Regulatory proteins, including histone deacetylase (HDT3) and argonaute 1, were more abundant after the start of induction treatment with 2,4-D, suggesting their role in 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, hormone response, and SAM metabolism. Our findings emphasize the modulation of the proteome at different stages during embryogenic callus initiation and identify regulatory proteins that might be involved with the activation of this process.

Project description:Sugarcane (Saccharum spp.) is one of the most important crops for sugar, biofuels and bioenergy production, becoming an important commodity in the world agricultural market for more than 100 countries. In this study, label-free quantitative proteomics and phosphoproteomics analyses were performed to investigate signaling events related to somatic embryo maturation and differentiation in sugarcane. Embryogenic callus (EC) at the multiplication (EC0) and after 14 days (EC14) under maturation were compared. A total of 251 phosphoproteins and 700 proteins were differentially regulated and accumulated, respectively, in the EC14/EC0 comparison. Metabolic pathways analysis showed that proteins and phosphoproteins are enriched in lysine degradation and starch/sucrose metabolism during multiplication, while differentiation of somatic embryo involves the regulation of energetic metabolism, including TCA cycle, oxidative phosphorylation, and carbon metabolism. Multiplication-related phosphoproteins were mainly associated to abscisic acid responses and transcriptional regulation comprising the TOPLESS (TPL), SNF1 kinase homolog 10 (KIN10), SEUSS (SEU), and LEUNIG_HOMOLOG (LUH) proteins. In maturation-related phosphoproteins, the phosphorylation of the proteins light harvesting complex photosystem ii, CURVATURE THYLAKOID 1B, vacuolar proton ATPase A1 and phytochrome interacting factor 3-LIKE 5 is associated to bioenergetic metabolism and carbon fixation. Motif analysis revealed 15 phosphorylation motifs, of which the [D-pS / T-x-D] motif was unique for the phosphopeptides identified during somatic embryo differentiation. Co-expression network analysis of proteins and phosphoproteins revealed the interaction between SNF1-related protein kinase 2 (SnRK2), abscisic acid responsive elements-binding factor 2 (ABF2), and KIN10, indicating the role of these proteins in the embryogenic competence in EC0. The interactions between ubiquitin-conjugating enzyme 5, ubiquitin-conjugating enzyme 35, small ubiquitin-like modifier 1, and histone deacetylase 1 may be involved in post-translational protein modification during the embryo maturation stage. argonaute 1 (AGO1) also interacts with POLTERGEIST (POL) and may integrate gene silencing and regulation of meristem identity during somatic embryo development. These results reveal novel dynamics of protein regulation in somatic embryogenesis and identify new potential players in somatic embryo differentiation and their phosphosites.

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Early regulatory networks driving somatic embryogenesis in Saccharum spp. L. revealed by time-resolved proteomics

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