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Global landscape of protein phosphorylation during plant regeneration initiation in cotton (Gossypium hirsutum L.)

BMC Biology volume 23, Article number: 116 (2025) Cite this article

Abstract

Background

Phosphorylation is one of the most common post-translational modifications and is central to many cellular signaling events; however, little is currently known about the phosphorylation landscape during somatic embryogenesis (SE) for plant regeneration.

Results

Here, we systematically analyzed the phosphoproteomic profile of three typical developmentally staged cultures of SE, non-embryogenic calli (NEC), primary embryogenic calli (PEC), and globular embryos (GE), in cotton (Gossypium hirsutum L.), the pioneer crop for genetic biotechnology applications. Our data revealed a total of 6301 quantifiable phosphorylation sites in 2627 quantifiable phosphoproteins from 5548 modified peptides, of which 1105 phosphoproteins (2147 sites) were differentially phosphorylated. Functional enrichment analyses revealed that differentially regulated phosphoproteins (DRPPs) were significantly enriched in DNA mismatch repair and peroxisome during callus embryogenic differentiation (PEC vs. NEC) and somatic embryo initiation (GE vs. PEC), respectively. Notably, six dynamic trajectory patterns of DRPP enrichment were observed. In addition, preferentially activated DRPPs with specific phosphorylation patterns were identified at different developmental stages. These DRPPs were mainly involved in hormone-responsive and photosystem events during initiation of plant regeneration.

Conclusions

Overall, this study identified a series of potential phosphoproteins responsible for SE trans-differentiation and plant regeneration, providing a valuable resource and molecular basis for understanding the regulatory pathways underlying cell totipotency at the post-translational modification level.

Background

Post-translational modifications (PTMs) are central to many cell signaling events [1], which can occur at any stage of the protein life cycle and play a key role in the functional proteome. Phosphorylation is one of the most common PTMs, as there are more than a thousand kinase genes that account for 3–4% of functional plant genes [2]. In response to external environmental stimuli and hormone signal transduction, protein modifications, especially phosphorylation, are controlled by a complex array of mechanisms that further regulate important biological processes in organisms by regulating protein activity and protein–protein interactions (PPIs). Given the importance of phosphorylation, identifying novel phosphorylated proteins and phosphorylation sites and exploring the functional roles of these phosphorylated proteins has generated great interest. Currently, phosphoprotein analyses in the oilseed rape seed filling process [3], soybean root hair interactions with Bradyrhizobia [4], abscisic acid regulation of rice leaves [5, 6], phosphoprotein analysis in cotton tissues [7,8,9], and the identification of phosphoproteins in several tissues of Arabidopsis thaliana [10] have been reported. However, much remains to be elucidated in the field of plant cell totipotency, which is why the question of how a single cell becomes a whole plant is currently ranked as one of the 25 most important questions in scientific research [11,12,13,14].

The classical example of plant cell totipotency, somatic embryogenesis (SE), is an effective tool for plant species propagation and genetic improvement and is a useful model for studying the regulatory network of embryogenic development and plant regeneration in vitro [15,16,17]. Furthermore, evidence that regenerable whole plants are derived from differentiated cells cultured in vitro clearly demonstrates the plasticity of plant cells [18,19,20]. In previous studies, many genes associated with plant SE have been identified in large-scale transcriptomes, proteomes, and epigenomes [21,22,23,24,25,26]. In recent years, a few typical phosphorylated proteins associated with SE have been identified in several species, including casein kinase I (CKI) [27], somatic embryogenesis receptor-like kinase (SERK) [28], mitogen-activated protein kinase (MAPK) [29], late embryogenesis abundant protein (LEA) [30, 31], ABA-responsive element binding factor 1 (ABF1) [31], SNF1 kinase homolog 10 (KIN10) [32], and histidine kinase (HK) [33]. However, the landscape of phosphorylation sites and phosphoproteins associated with SE in plant regeneration remains unknown. Therefore, research on the molecular system surrounding phosphorylation in plant regeneration has important fundamental and practical implications for plant cell engineering and asexual lineage biotechnology in crop breeding and for improving agricultural productivity.

Phosphoproteomics, as highlighted in the recent study [34], has emerged as a powerful tool to unravel the complex regulatory networks underlying plant development. In this study, high-throughput phosphoproteomics of non-embryogenic calli (NEC), primary embryogenic calli (PEC), and globular embryos (GE) during SE were systematically analyzed in cotton (Gossypium hirsutum L.), a pioneer crop for plant biotechnology in cell and genetic engineering, which commonly achieves plant regeneration through the SE pathway. The aim of this study was to reveal a series of characteristic phosphosites and phosphoproteins involved in SE and novel phosphoprotein networks involved in this process. This study uncovers fundamentally important findings and practically valuable gene resources for plant regeneration, offering promising avenues for advancements in plant cell engineering and somatic asexual reproduction applications.

Results

Construction of the phosphoproteomic landscape in plant regeneration initiation

To identify the post-translational events associated with plant regeneration initiation, we sampled the critical representative periods of NEC, PEC, and GE for a global analysis of the phosphorylation modification panel (Fig. 1A). In this study, to enhance the mining of phosphoproteomics data, we employed a series of cutting-edge technologies, including tandem mass tag (TMT) labeling, high-performance liquid chromatography (HPLC) classification, and enrichment of phosphorylated peptides. Quantitative protein proteomics based on mass spectrometry (MS) was then performed to determine the phosphorylation profile of proteins during SE. To verify the dimensionality of the dataset, the data were converted into a new set of variables using principal component analysis, which summarized the data features (Fig. 1B). The pairwise Pearson’s correlation coefficients displayed sufficient reproducibility (Fig. 1C). Of these spectra, 5548 modified peptides of 6805 peptides were detected (Table 1), and the average peptide mass error was < 10 ppm (Additional file 1: Fig. S1A), indicating the high mass accuracy of the MS data. The lengths of most of the identified peptides and their modification site distributions are shown in Additional file 1: Fig. S1B and S1C. The above data indicated that the MS detection results of the phosphoproteome were of high quality and accuracy, suggesting that the samples met the required standards. Two representative tandem mass spectrometry (MS/MS) spectra of the phosphorylated peptides are shown as examples in Additional file 1: Fig. S1D. Detailed information on the proteins and their phosphorylation sites/peptides is shown in Additional file 1: Table S1 and S2 (localization probability > 0.75). We identified a total of 6301 quantifiable phosphorylation sites in 2627 proteins (Table 1). These results provide a comprehensive phosphoproteomic dataset for future studies on plant regeneration.

Fig. 1
figure 1

Experimental strategy of the phosphorylation modification panel in three representative periods during SE. A Workflow of phosphorylated proteome experiments in three representative periods of cotton SE (non-embryogenic calli, NEC, cultured for 5-6 weeks; primary embryogenic calli, PEC, cultured for additional 2-4 weeks; globular embryos, GE, cultured for additional 4-6 weeks). Scale bar = 1mm. B Principal component analysis (PCA) of the three staged samples. C Mass delta of all identified phosphopeptides. Three biological replicates are provided for each sample stage (NEC, PEC, and GE)

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Table 1 Tandem mass spectrometry database search analysis summary
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Identification of phosphorylation sites and phosphoproteins

Phosphorylation sites are footprints of kinase activity. Investigating the amino acid context around phosphorylation sites provides valuable information for predicting these sites in unidentified proteins. Conserved phosphate-site motifs also contribute to the association of substrate phosphoproteins with their kinases. To understand the properties of phosphorylation and identify specific amino acids adjacent to phosphosites, we examined the amino acid sequences flanking phosphosites by generating a heatmap (Fig. 2A; Additional file 1: Fig. S2A). Substantial bias in amino acid distribution was observed from the − 6 to + 6 positions around phosphorylation sites. Seven amino acid residues, aspartic acid (D), glutamine (E), glycine (G), histidine (H), proline (P), arginine (R), and serine (S), were over-represented in regions surrounding the phosphorylation sites (Fig. 2A). To assess phosphosite conservation, we used WebLogo to produce sequence logos (Fig. 2B) typical of multiple sequence alignments between all phosphosites and differential ones. This information can facilitate the prediction of phosphorylation sites in unidentified proteins.

Fig. 2
figure 2

Analysis of the identified phosphosites. A Heatmap of the amino acid sequences flanking phosphosites. The central S refers to the phosphorylated serine. B Enrichment analysis of phosphoproteins motifs. The height of each letter corresponds to the frequency of that amino acid residue in that position. The central S refers to the phosphorylated serine. C Proportion of single phosphosites and multi-phosphosites. D Proportion of amino acids in the identified phosphopeptides

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Within a specific phosphorylated protein, the number of phosphorylation sites varies widely. While only a few phosphorylation sites were detected on most proteins, a minority of proteins had more than six phosphorylation sites (Fig. 2C; Additional file 1: Table S1). The maximum number of phosphorylation sites was 37 in A0A1U8MXR8, which encodes a PWI domain-containing protein (Additional file 1: Table S1). The percentage of phosphoproteins with different numbers of phosphorylation sites is shown in Fig. 2C, where 43.88 and 25.72% had one or two phosphorylation sites, respectively. Notably, approximately 9.07% (300) of the identified phosphorylated proteins contained more than six phosphorylation sites (Additional file 1: Table S1). At the level of phosphorylated amino acid residues, of the 8475 identified phosphorylation sites, 88.8% (7528) were phosphorylated on serine (pSer), 8.9% (765) on threonine (pThr), and 2.2% (192) on tyrosine (pTyr) (Fig. 2D), suggesting a critical role of serine phosphorylation in regulating protein function during plant regeneration.

In this study, we used WoLF PSORT to predict the distribution of identified phosphoproteins and differentially regulated phosphoproteins (DRPPs) in various subcellular compartments. As shown in Additional file 1: Fig. S2B, most of the phosphoproteins were in the nucleus (51.42%), chloroplast (17.49%), cytoplasm (14.23%), and plasma membrane (10.16%), suggesting their involvement in nuclear signaling, photosynthesis, cellular metabolism, and membrane-associated processes. Other cellular compartments, including the mitochondria, endoplasmic reticulum, Golgi apparatus, and vacuolar membrane, collectively accounted for 6.70% of the phosphoproteins (Additional file 1: Fig. S2B). We also examined the subcellular distribution of the three comparison groups during SE (Additional file 1: Fig. S2B), which indicated a conserved distribution pattern across tissues.

Identification of DRPPs during SE

In the current study, 3309 phosphoproteins (8475 phosphorylation sites) were identified, of which 2627 phosphorylated proteins (6301 phosphorylation sites) provided quantitative information (Table 1). To gain in-depth insight into their phosphorylation patterns, we compared the data of the three samples pairwise according to their phosphorylation intensity. A fold change exceeding 2 indicated significant upregulation, whereas less than 0.5 indicated significant downregulation. In the three comparison groups, PEC vs. NEC, GE vs. PEC, and GE vs. NEC, 1405 (695 DRPPs), 306 (226 DRPPs) and 1814 (953 DRPPs) phosphorylation sites, respectively, were identified. The number of upregulated and downregulated DRPPs is shown in Table 2, and further protein description details are shown in Additional file 1: Table S3. Our comparative analysis of NEC, PEC, and GE samples revealed 1105 differentially regulated phosphoproteins (DRPPs) with 2147 phosphorylation sites. These DRPPs showed distinct patterns of upregulation and downregulation during SE, indicating dynamic changes in phosphorylation status associated with plant regeneration.

Table 2 Summary of differentially regulated phosphoproteins
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To visualize the differentially regulated phosphoproteins, we generated Venn diagrams (Fig. 3A, B) to compare the unique and overlapping DRPPs between PEC vs. NEC, GE vs. PEC, and GE vs. NEC groups. This analysis revealed specific and common phosphorylation events during SE. The results showed that the PEC vs. NEC, GE vs. PEC, and GE vs. NEC groups specifically enriched 111 DRPPs (252 sites), 36 DRPPs (59 sites), and 266 DRPPs (510 sites), respectively. Sixty-four common DRPPs were simultaneously involved in NEC, PEC, and GE processes.

Fig. 3
figure 3

Venn and GO enrichment analyses of differential phosphoproteins. A Venn diagram of the differential phosphoproteins in the three comparison groups (PEC vs. NEC, GE vs. PEC, and GE vs. NEC) at the protein level. B Venn diagram of the differential phosphoproteins in the three comparison groups (PEC vs. NEC, GE vs. PEC, and GE vs. NEC) at the phosphosite level. C–E GO enrichment analysis of the differential phosphoproteins in PEC vs. NEC, GE vs. PEC, and GE vs. NEC

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To gain insights into the biological processes and pathways affected by these DRPPs, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses (Additional file 1: Table S3). GO enrichment analysis revealed that DRPPs were significantly enriched in various cellular compartments, molecular functions, and biological processes. For example, in the PEC vs. NEC comparison group (representing callus embryogenic differentiation), DRPPs were enriched in biological processes related to metabolism of nucleic acids and various compounds (Fig. 3C). In contrast, during somatic embryo initiation (GE vs. PEC), DRPPs were enriched in response to inorganic substance and chloride transport (Fig. 3D). KEGG enrichment analysis further supported the involvement of DRPPs in specific metabolic and signaling pathways. In the PEC vs. NEC group, pathways such as DNA replication and mismatch repair were enriched (Additional file 1: Fig. S3), suggesting their role in maintaining genomic integrity during early stages of SE. During somatic embryo initiation (GE vs. PEC), DRPPs were enriched in the peroxisome and photosynthesis-related pathways (Additional file 1: Fig. S3), indicating potential roles in metabolic adaptation and energy production necessary for early embryo development.

Dynamic trajectory analysis of DRPPs during plant regeneration initiation

We traced the trajectories of phosphorylated protein enrichment during SE and performed a hierarchical clustering analysis (HCA), as shown in Fig. 4. Six dynamic patterns were observed at the different developmental stages, indicating complex changes involving multiple pathways during plant regeneration in vitro; information on the DRPPs and sites in each cluster is presented in Additional file 1: Table S4.

Fig. 4
figure 4

Hierarchical Clustering Analysis (HCA) of the differentially phosphorylated proteins during SE. Cluster identification and number of profiles included in each cluster are indicated on the left. The color of the line indicates the affiliation of the protein to the current class. For the heat map, the x-axis represents the samples, the y-axis represents the different phosphoproteins, and the color indicates the relative abundance of each protein in the sample. The top 2 most significantly enriched items from the GO and KEGG enrichment analyses are shown to the right of the corresponding cluster enrichment pattern clustering graph. GO_CC, cellular component; GO_BF, biological process; GO_MF, molecular function

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For PEC, an important period for regeneration initiation, clusters 2, 4, and 5 exhibited a characteristic PEC pattern (Fig. 4). Of the six dynamic patterns, the second cluster was the largest with 666 phosphorylation sites (310 DRPPs). The cluster exhibiting a surge pattern from NEC to PEC and GE suggests that this class of proteins plays an equally important role in the PEC and GE periods during the SE. Additionally, 32 phosphorylation sites were detected in cluster 2 for serine/arginine repetitive matrix protein 1-like (protein ID: A0A1U8MXR8), which is one of the most strongly phosphorylated proteins in the phosphoproteome. The complexity and extent of phosphorylation of this protein suggest that it may be involved in intricate regulatory networks that contribute to the precise control of splicing events and gene expression (Additional file 1: Fig. S4). Furthermore, 211 phosphorylation sites (110 DRPPs) in cluster 5 exhibited a remarkable enrichment pattern in PEC, and KEGG enrichment results indicated that the cluster was significantly enriched for plant hormone signal transduction, mRNA surveillance pathways, and ABC transporters (Fig. 4; Additional file 1: Fig. S4D). Conversely, cluster 4 showed a linear declining pattern from NEC to PEC, enriching 294 phosphorylation sites (191 DRPPs) and mainly localizing to the plasma membrane and cell membrane. In addition, cluster 1 showed a linear upregulation pattern from PEC to GE and was mainly localized in chloroplasts and plastids. Importantly, 23 phosphosites (16 DRPPs) exhibited upregulation followed by downregulation in cluster 5, and five phosphosites (5 DRPPs) exhibited downregulation followed by upregulation in clusters 1 and 4 (Table 3; fold change > 2), all of which showed significant specificity in the PEC period.

Table 3 List of differentially regulated phosphoproteins with significant characteristics during plant regeneration initiation
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In addition, GE, compared to the significantly enriched NEC and PEC phosphoprotein sites of clusters 1, 3, and 6, showed two types of regulation patterns overall. In the first type, 241 phosphosites (172 DRPPs) of cluster 1 with an upregulated pattern were significantly enriched in the chloroplasts of the GO cellular component term, indicating that photosystem-related DRPPs may be involved in GE formation (Fig. 4; Additional file 1: Fig. S4A). Furthermore, 525 phosphosites (321 DRPPs) were enriched in cluster 3 with a linear trend and may be involved in biological processes such as gene expression and cellular nitrogen compound metabolism (Fig. 4; Additional file 1: Fig. S4C). Regarding the second type, the 207 phosphosites (147 DRPPs) enriched in cluster 6 with a downregulated pattern may have transmembrane transport activity (Fig. 4; Additional file 1: Fig. S4B).

Taken together, the proteins involved in phytohormone responses and photosystem events during the initiation of plant regeneration were identified in this analysis as potential molecular markers in future crop cell and gene engineering. And the identification of specific DRPPs and their phosphorylation patterns provide insights into the molecular basis of SE development. In addition, the hierarchical clustering analysis identified distinct dynamic patterns of DRPPs during SE, indicating complex changes involving multiple pathways. This observation underscores the need for further integrative multiomics approaches to fully unravel the regulatory networks governing plant regeneration.

PPI networks among phosphoproteins during SE

To establish PPI networks for the NEC-, PEC-, and GE-related phosphoproteins, we extracted the interaction relationship of DRPPs by aligning with the STRING (v.11.0) protein interaction network database according to the confidence score > 0.7 (high confidence). The complete PPI networks of DRPPs during SE are shown in Fig. 5. The PPI network of the PEC vs. NEC terms had two clusters containing 79 DRPPs, in which the phosphorylation sites of the splicing factor 3B subunit 1-like proteins (POPTR_0017s07760.1) showed bidirectional regulation (Fig. 5A; Additional file 1: Table S5). Notably, this protein potentially interacted with 11 DRPPs, of which 9 were located in the cell nucleus (Fig. 5A; Additional file 1: Tables S3 and S5). Therefore, we speculated that splicing factor 3 B subunit 1-like might have been simultaneously influenced by multiple regulatory factors, exhibiting competitive effects and resulting in the upregulation or downregulation of the phosphorylation site under different conditions. Moreover, 25 DRPPs were enriched in the spliceosome pathway (ghi03040) in terms of PEC vs. NEC and were the most abundant PPI clusters. Eleven clusters in the GE vs. PEC term comprised 27 DRPPs (Fig. 5B; Additional file 1: Table S5). In the GE vs. NEC comparison, 16 clusters and 125 DRPPs were identified (Fig. 5C; Additional file 1: Table S5).

Fig. 5
figure 5

Protein–protein interaction (PPI) network analysis. PPI network analysis of the comparison groups. PEC vs. NEC (A), GE vs. PEC (B), and GE vs. NEC (C). Gh_D13G1800, cell division cycle 5-like protein (CDC5); Gh_D10G2059, pyruvate kinase (PK); Gh_A09G0792, heat shock 70 kDa protein (HSP70); Gh_D13G0352, serine/threonine-protein kinase SRK2B (SRK2B)

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Discussion

Plants possess a remarkable capacity for somatic cell totipotency and regeneration [35,36,37,38]. However, plant biotechnological applications in cell and genetic engineering and crop breeding are severely limited by the low frequency of SE and high rate of malformed embryos in regeneration. Therefore, an in-depth study of the molecular basis and complex regulatory networks of SE is important to achieve efficient regeneration and accelerate the crop breeding process.

Phosphorylation is a major post-translational modification and plays a critical role in various cellular processes, including signal transduction, gene expression regulation, and protein activity modulation [39]. In the context of SE, phosphorylation is likely to be involved in key signaling events and regulatory mechanisms underlying embryogenic differentiation and embryo development. Despite its potential importance, research on phosphorylation in the field of plant regeneration remains limited.

In this study, we aimed to investigate the significance of phosphorylation in plant regeneration and shed light on the poorly understood landscape of proteins phosphorylated during SE. We characterized the phosphoproteomic profile of three typical developmental stages of SE and identified DRPPs as potential targets for improving the efficiency of SE. Our findings, in line with the observations made by Cruz-Mireles et al., suggest that phosphorylation plays a pivotal role in regulating key signaling pathways and gene expression [34]. While our previous study explored integrated proteomic and phosphoproteomic analyses [40, 41], this work uniquely dissects phosphorylation dynamics in SE trans-differentiation and plant regeneration. Through our research, we hope to contribute to the understanding of phosphorylation-mediated processes during SE and provide valuable insights and genetic resources for enhancing plant regeneration and advancing crop breeding strategies.

Protein kinases involved in plant regeneration initiation

Many phosphoproteins can be modified by different types of protein kinases, which occupy an important position in plant functional genes and regulate a variety of processes, including metabolism and hormonal responses [42]. In the present study, protein kinases were identified at all three SE stages, similar to a previous report on early rice embryo germination [43], highlighting the importance of protein phosphorylation in regulating plant regeneration [44]. In the HCA analysis, we screened five protein kinases (seven phosphorylation sites) in cluster 5 and eight protein kinases (eight phosphorylation sites) in cluster 1, which exhibited an upregulated trend during the PEC and GE periods, respectively (Table 4). The protein kinases in clusters 5 and 1, which exhibited a remarkable enrichment pattern in the PEC and GE stages, respectively, suggested their potential involvement in stage-specific signaling events and regulatory processes.

Table 4 Protein kinases and transcription-related differentially regulated phosphoproteins involved in plant regeneration initiation
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In this study, the significantly upregulated mitogen-activated protein kinase (MAPK) in the PEC vs. NEC group of cluster 5 suggests that MAPK is involved in the signal transduction process in plant embryogenic differentiation. In particular, MKK6 (G8AA64) was differentially regulated in both the PEC vs. NEC and GE vs. PEC groups, exhibiting a peak characteristic of phosphorylated proteins, suggesting that it may be involved in signaling pathways, such as the MAPK cascade, which plays a crucial role in plant regeneration initiation. This finding is consistent with the previous study in which phosphorylation event was controlled by major regulators, such as MAPKs [34], and recent multiomics studies, which have highlighted the importance of MAPK cascades in regulating plant SE development [24, 45].

Additionally, we identified three DRPPs (A0A1U8JLE0, A0A1U8JP86, and A0A1U8K8P6) with conserved protein kinase domains and serine/threonine kinase activities in cluster 1. These findings suggest their potential involvement in novel and uncharacterized signaling pathways related to SE or plant regeneration. In addition, the loss-of-function of Arabidopsis histidine kinase (AHK2 and AHK3) genes induces a typical phenotype of impairment in cytokinin signaling, affecting Arabidopsis callus formation and the development of plant organs such as roots and leaves [46]. The specific phosphorylation-enriched pattern of histidine kinase (A0A1U8KR14) in the GE stage suggests a key role in plant regeneration. Furthermore, the first plant inositol 1,3,4-trisphosphate 5/6-kinase (AtITPK1) is involved in Arabidopsis photomorphogenesis [47], seed coat development [48], and phytate biosynthesis during maize seed development [49] but has not been reported in plant SE. The specific phosphorylation pattern of ITPK (A0A1U8KD24) in our study provides a basis for studying its function in regulating plant regeneration.

Transcription-related phosphoproteins involved in plant regeneration initiation

In previous studies, numerous transcription factors associated with plant SE have been identified in large-scale transcriptomes [21, 50]. However, unlike the genome, the differential dynamic processes of protein abundance, structure, stability, subcellular localization, and continuous interactions with other biological macromolecules are regulated by post-translational modifications [7]. We screened six transcription-related DRPPs (eight phosphorylation sites) in cluster 5 and six transcription-related DRPPs (nine phosphorylation sites) in cluster 1 that exhibited upregulated trends during the PEC and GE periods, respectively (Table 4). Three of these uncharacterized transcription factors, A0A1U8MT06, A0A1U8PDX7, and A0A1U8KL00, belong to the BREVIS RADIX [51], IIS [52], and MYC/MYB [53] gene families, respectively; however, their molecular biological functions are unknown. Their differential expression patterns and potential regulatory functions make them intriguing candidates for further investigation and characterization to elucidate the molecular mechanisms underlying embryogenesis. Taken together, these findings indicate that the phosphorylation-mediated regulation of transcription factors provides a novel perspective and highlights the involvement of these proteins as potential key players in the SE process.

Furthermore, two transcription factors in cluster 3 (A0A1U8PE78 and A0A1U8MKC1) were linearly upregulated during SE (Table 4). This upregulation indicates that these transcription factors play essential roles in driving the expression of genes necessary for embryo formation, thus contributing to the establishment and progression of the embryogenic developmental program. WRKY proteins play an important role in plant development and stress responses, and their transcriptional activity is dependent mainly on MPK-mediated phosphorylation modifications [54]; in the context of our findings, the upregulated trend of probable WRKY transcription factor 31 (A0A1U8PE78) during SE suggests its potential involvement in the regulatory processes underlying plant regeneration initiation. Therefore, we speculate that WRKY31 phosphorylation modulates its transcriptional activity and interaction with other regulatory proteins, thus influencing the expression of downstream target genes involved in embryogenesis and stress responses. Considering these findings, future research on WRKY proteins should aim to elucidate their specific functions and regulatory mechanisms in plant regeneration, as investigating their interactions with other signaling components such as MPKs and their involvement in downstream gene expression networks would be valuable.

In addition, the upregulated trend of the transcription factor IWS1-like (A0A1U8MKC1) during SE suggests its potential role in regulating processes associated with plant regeneration initiation. The IWS1 protein has been reported to be involved in the post-recruitment of RNAPII-mediated transcription to facilitate mRNA export, suggesting an important role for AtIWS1 in the induction of gene expression by plant hormones and regulation of essential plant growth and developmental processes [55]. In addition, yeast IWS1/SPN1 recruits the transcription elongation factor SPT6 and the SWI/SNF chromatin remodeling complex during transcriptional activation [56]. Interestingly, the transcription elongation factor SPT6-like isoform X2 (A0A1U8L372) of cluster 5 was identified during SE in our study, suggesting its importance in promoting embryogenic differentiation. A previous report indicated that the heterodimeric complex SPT4/SPT5 plays a role as a transcription elongation factor in Arabidopsis, regulating transcription during the elongation phase and influencing the expression of certain auxin-related genes [57]. Based on these findings, we speculate that the transcription factor IWS1 and transcription elongation factor SPT6 identified in this study may interact to modulate gene expression during SE, potentially influencing the expression of auxin-related genes and other developmental processes. However, further investigations are needed to elucidate the molecular mechanisms underlying the roles of these factors in SE and their potential interactions with hormone signaling pathways and other regulatory networks.

The specific hormones and related genes in phosphorylation-mediated plant regeneration

Our findings highlight the importance of phosphorylation in regulating hormone signaling during plant regeneration. By analyzing the phosphoproteome, we identified several key proteins and phosphorylation sites involved in auxin and abscisic acid signaling, which are well-known regulators of plant growth and development. For instance, the phosphorylation of auxin response factors (ARFs) suggests that auxin signaling is dynamically regulated during the NEC, PEC, and GE stages. Previous studies have shown that auxin promotes cell dedifferentiation and reprogramming during plant regeneration [58, 59]. Therefore, our findings provide additional evidence for the role of phosphorylation in modulating auxin-mediated cell fate transitions.

Similarly, the differential phosphorylation of proteins involved in abscisic acid signaling (ABA responsive element binding factors, ABFs) suggests that this hormone also participates in phosphorylation process during plant regeneration. Abscisic acid plays a pivotal role in regulating various physiological processes, including stress tolerance and embryogenesis [31, 60]. Our study revealed phosphorylation sites on abscisic acid response regulators, which may represent important regulatory switches in abscisic acid signaling during SE.

Phosphoproteomic resource for understanding plant cell embryogenic differentiation and regeneration

The identified DRPPs are likely to be key regulators of SE and can serve as potential targets for improving SE efficiency in cotton and other plant species. For instance, the upregulation of CKI phosphorylation sites during SE suggests that these kinases are activated to promote cotton regeneration development. Similarly, the differential regulation of proteins involved in DNA repair and peroxisome biogenesis may be crucial for ensuring genomic stability and metabolic homeostasis during regeneration.

Furthermore, the dynamic trajectory analysis revealed six distinct patterns of DRPP enrichment during SE (Fig. 4), indicating complex and intricate changes in protein phosphorylation networks that underlie plant regeneration (Fig. 6). These patterns highlight the presence of multiple regulatory mechanisms operating at different stages of SE, each contributing to the successful initiation and progression of plant regeneration.

Fig. 6
figure 6

Model diagram of phosphorylated protein regulation in cotton somatic embryogenesis

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Overall, our study not only provides a comprehensive phosphoproteomic resource for cotton SE but also sheds light on the biological significance of protein phosphorylation in regulating plant regeneration process. Further research is needed to validate the functional roles of these DRPPs and phosphorylation sites in plant regeneration. Functional studies using mutants, CRISPR/Cas9 genome editing, and phospho-site-specific antibodies will be valuable in elucidating the precise mechanisms by which phosphorylation regulates hormone signaling and cell fate decisions during plant regeneration.

Conclusions

This study provides a comprehensive phosphoproteomic landscape of cotton somatic embryogenesis, identifying 6301 phosphorylation sites in 2627 proteins. The results reveal key phosphoproteins and phosphorylation events regulating plant regeneration initiation. Dynamic changes in phosphorylation patterns suggest their roles in hormone signaling during embryogenic differentiation. This work contributes valuable insights into the molecular mechanisms underlying plant cell totipotency and regeneration, offering potential targets for crop improvement.

Methods

Plant materials and culture conditions

In vitro tissue culture of upland cotton (Gossypium hirsutum cv. YZ-1) hypocotyl (HY) explants was carried out based on a procedure described in our previous studies [25, 61]. Briefly, 0.5–1 cm HYs were induced by dedifferentiation on Murashige and Skoog with B5 (MSB) medium containing 0.45 µmol·L-1 2,4-dichlorophenoxyacetic acid and 0.46 µmol·L-1 kinetin to form NEC. After approximately 5–6 weeks, the cells were subcultured on fresh phytohormone-free MSB medium to enter the redifferentiation stage. After a further 2–4 weeks of growth, the somatic to embryogenic transformation proceeded to the induction stage of PEC development. Subsequently, small millet-shaped mature embryogenic callus masses were harvested for further proliferation and purification and gradually differentiated into hard granular early GE. Three representative periods of cotton SE (NEC, PEC, and GE) were collected and immediately snap-frozen in liquid nitrogen and stored at − 80 ℃ for protein extraction. Each staged sample was prepared in triplicate.

Protein extraction and tryptic digestion

To prepare the protein samples, the three period samples were ground into powder using liquid nitrogen and transferred to 5-mL centrifuge tubes. The powder was then treated with four volumes of lysis buffer (containing 8 M urea, 1% Triton-100, 10 mM dithiothreitol, and 1% Protease Inhibitor Cocktail). This was followed by sonication using a high-intensity ultrasonic processor (Scientz, Ningbo, China) on ice. Remaining debris was removed by centrifugation. Subsequently, the protein was precipitated with cold 20% trichloroacetic acid and washed three times with cold acetone. The protein was then re-dissolved in 8 M urea and its concentration determined using a BCA kit (Beyotime Biotechnology, Shanghai, China). For digestion, the protein solution was alkylated and reduced, followed by trypsin digestion at two different ratios overnight and for 4 h, respectively.

TMT labeling and HPLC fractionation

TMT labeling and HPLC fractionation were carried out as described in our previous study [61].

Affinity enrichment

The peptide mixtures were incubated with immobilized metal affinity chromatography (IMAC) microspheres suspended in a loading buffer containing 50% acetonitrile and 6% trifluoroacetic acid. After centrifugation, the phosphopeptide-rich IMAC microspheres were collected, and the supernatant discarded. To remove nonspecific peptide adsorption, the microspheres were washed successively with two buffers, 50% acetonitrile/6% trifluoroacetic acid and then 30% acetonitrile/0.1% trifluoroacetic acid. Elution of the phosphopeptides was achieved by adding an elution buffer containing 10% NH4OH, followed by vibration. The supernatant containing the phosphopeptides was collected, lyophilized, and then prepared for liquid chromatography (LC)-MS/MS analysis.

LC–MS/MS analysis and database search

The gradient elution on the EASY-nLC 1000 UPLC system (Thermo Fisher Scientific, Waltham, MA, USA) involved a stepwise increase in solvent B (0.1% formic acid in 98% acetonitrile), from 6 to 23% over 26 min, followed by a linear increase to 35% over 8 min, and then a rapid increase to 80% over 3 min. This composition was maintained for the final 3 min. The detailed procedures for MS/MS peptide analysis and database search have been previously reported by Guo et al. [61].

Subcellular localization prediction

We utilized the updated version of PSORT/PSORT II, known as WoLF PSORT subcellular localization prediction software (http://www.genscript.com/psort/wolf_psort.html), to predict the subcellular localization of eukaryotic sequences.

Motif analysis

Soft motif-x was employed to analyze the model of sequences, which encompassed amino acids at designated positions of modify-21-mers (inclusive of 10 amino acids upstream and downstream of the site), and phosphorylation was analyzed using modify-13-mers (comprising 6 amino acids upstream and downstream of the site). This analysis was conducted for all protein sequences, with the entire database of protein sequences serving as the background database parameters, while other settings were left at their default values.

HCA and GO, KEGG pathway analyses

The R package Mfuzz was utilized for the HCA of differentially phosphorylated sites based on relative phosphorylation intensity.

For Gene Ontology (GO) annotation, proteins were categorized into three groups: biological processes, cellular compartments, and molecular functions. A two-tailed Fisher’s exact test was conducted to assess the enrichment of differentially modified proteins within each category, comparing them to all identified proteins. GO terms with a corrected p-value less than 0.05 were considered statistically significant.

To identify enriched pathways, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database was utilized. Two-tailed Fisher’s exact test was applied to determine the enrichment of differentially modified proteins against all identified proteins. Pathways with a corrected p-value below 0.05 were considered statistically significant and were further classified into hierarchical categories based on the KEGG website.

PPI network analysis

The PPI network analysis was performed using the STRING software (version 10.5), applying a confidence score threshold of > 0.7. The resulting network was then visualized using Cytoscape software (version 3.6.1).

Data availability

All relevant data can be found and available within the manuscript and supplemental information. The proteomic data analyzed in this study have been submitted to PRIDE with accession number PXD062095. The phosphoproteomics data generated in this study have been submitted to PRIDE and the accession number is PXD061812.

Abbreviations

ABF:

Abscisic acid responsive element binding factor

ARF:

Auxin response factor

DRPP:

Differentially regulated phosphoprotein

GE:

Globular embryos

HCA:

Hierarchical clustering analysis

MAPK:

Mitogen-activated protein kinase

NEC:

Non-embryogenic calli

PEC:

Primary embryogenic calli

SE:

Somatic embryogenesis

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Acknowledgements

We are grateful for the supports with helpful research suggestions and discussions from Research Institute of Biology and Agriculture at USTB. And we thank for technical support provided by Jingjie Biotechnology Co. Ltd., Hangzhou, China.

Funding

This project was supported by Modern Agro-industry Technology Research System of Shandong Province (SDAIT-03–02), National Key Research and Development Program (2024YFD1200300), Taishan Scholar Talent Project from PRC (tsqn202312154), Shandong Province Excellent Youth Fund (ZR2023YQ022), and the National Natural Science Foundation of China (32372142).

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  1. Huihui Guo, Xiushan Qi and Li Zhang contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Tai’an, 271018, People’s Republic of China

    Huihui Guo, Xiushan Qi, Li Zhang, Haixia Guo, Fan Gao, Xindi Tian, Jianfei Wu, Tongtong Li, Tongdi Yan, Xiwang Cui, Jiawei Xu & Fanchang Zeng

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Contributions

F.C.Z. and H.H.G. conceived and designed the study. H.H.G., L.Z., and H.X.G. performed tissue sampling. H.H.G., L.Z., H.X.G., F.G., X.D.T., J.F.W., T.T.L., T.D.Y., and J.W.X. performed all morphological and molecular experiments. X.S.Q, X.W.C., H.H.G., H.X.G., and F.C.Z. analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.

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Additional file 1: Tables S1-S5 and Figures S1-S4. Table S1. Detailed information regarding phosphorylation sites. Table S2. Detailed information regarding phosphorylation peptides. Table S3. Differentially regulated phosphoproteins and phosphosites. Table S4. Annotation of differentially regulated phosphoprotein clusters for the hierarchical clustering analysis. Table S5. Information related to the protein–protein interaction analyses of the primary embryogenic calli vs. non-embryogenic calli, globular embryos vs. primary embryogenic calli, and globular embryos vs. non-embryogenic calli groups. Fig. S1. Quality control validation of the MS data. A Average phosphopeptide mass error. B Length distribution of all identified phosphopeptides. C Distribution of all identified phosphosites. D Representative MS/MS spectra of phosphopeptides from two proteins. Fig. S2. Analysis of the modification sites and subcellular localization. A Heatmap of the amino acid sequences flanking phosphosites. The central T refers to the phosphorylated threonine. B Subcellular localization of identified and differential phosphoproteins. Fig. S3. KEGG enrichment analysis of the differentially phosphorylated proteins. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of up- and down-regulated differentially phosphorylated proteins. Fig. S4. GO and KEGG enrichment plots of the differentially phosphorylated proteins in the HCA clusters. A–C Gene Ontology (GO) enrichment plots of the individual cluster. A GO-CC: cellular component. B GO-MF: molecular function. C GO-BF: biological process. D KEGG enrichment plots of the individual cluster.

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Guo, H., Qi, X., Zhang, L. et al. Global landscape of protein phosphorylation during plant regeneration initiation in cotton (Gossypium hirsutum L.). BMC Biol 23, 116 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12915-025-02218-7

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