Plant regeneration via somatic embryogenesis (SE) is a key step during genetic engineering. In the current study, integrated widely targeted metabolomics and RNA sequencing were performed to investigate the dynamic metabolic and transcriptional profiling of cotton SE. Our data revealed that a total of 581 metabolites were present in nonembryogenic staged calli (NEC), primary embryogenic calli (PEC), and initiation staged globular embryos (GE). Of the differentially accumulated metabolites (DAMs), nucleotides, and lipids were specifically accumulated during embryogenic differentiation, whereas flavones and hydroxycinnamoyl derivatives were accumulated during somatic embryo development. Additionally, metabolites related to purine metabolism were significantly enriched in PEC vs. NEC, whereas in GE vs. PEC, DAMs were remarkably associated with flavonoid biosynthesis. An association analysis of the metabolome and transcriptome data indicated that purine metabolism and flavonoid biosynthesis were co-mapped based on the Kyoto encyclopedia of genes and genomes (KEGG) database. Moreover, purine metabolism-related genes associated with signal recognition, transcription, stress, and lipid binding were significantly upregulated. Moreover, several classic somatic embryogenesis (SE) genes were highly correlated with their corresponding metabolites that were involved in purine metabolism and flavonoid biosynthesis. The current study identified a series of potential metabolites and corresponding genes responsible for SE transdifferentiation, which provides a valuable foundation for a deeper understanding of the regulatory mechanisms underlying cell totipotency at the molecular and biochemical levels.

The somatic embryogenesis (SE) process of plants, as one of the typical responses to abiotic stresses with hormone, occurs through the dynamic expression of different proteins that constitute a complex regulatory network in biological activities and promotes plant totipotency. Plant SE includes two critical stages: primary embryogenic calli redifferentiation and somatic embryos development initiation, which leads to totipotency. The isobaric labels tandem mass tags (TMT) large-scale and quantitative proteomics technique was used to identify the dynamic protein expression changes in nonembryogenic calli (NEC), primary embryogenic calli (PEC) and globular embryos (GEs) of cotton. A total of 9369 proteins (6730 quantified) were identified; 805, 295 and 1242 differentially accumulated proteins (DAPs) were identified in PEC versus NEC, GEs versus PEC and GEs versus NEC, respectively. Eight hundred and five differentially abundant proteins were identified, 309 of which were upregulated and 496 down regulated in PEC compared with NEC. Of the 295 DAPs identified between GEs and PEC, 174 and 121 proteins were up- and down regulated, respectively. Of 1242 differentially abundant proteins, 584 and 658 proteins were up- and down regulated, respectively, in GEs versus NEC. We have also complemented the authenticity and accuracy of the proteomic analysis. Systematic analysis indicated that peroxidase, photosynthesis, environment stresses response processes, nitrogen metabolism, phytohormone response/signal transduction, transcription/posttranscription and modification were involved in somatic embryogenesis. The results generated in this study demonstrate a proteomic molecular basis and provide a valuable foundation for further investigation of the roles of DAPs in the process of SE transdifferentiation during cotton totipotency.

As a notable illustration of totipotency and plant regeneration, somatic embryogenesis (SE) is the developmental reprogramming of somatic cells toward the embryogenesis pathway, the key step for genetic engineering. Investigations examining the totipotency process are of great fundamental and practical importance in crop biotechnology. However, high-frequency regeneration of cotton via SE has been limited due to genotype-dependent response. The molecular basis deciphering SE genotype recalcitrance remains largely unexplored in cotton. In the current study, to comprehensively investigate the dynamic transcriptional profiling and gene regulatory patterns involved in SE process, a genome-wide RNA sequencing analysis was performed in two cotton genotypes with distinct embryogenic abilities, the highly embryogenic genotype Yuzao 1 (YZ) and the recalcitrant genotype Lumian 1 (LM). Three typical developmental staged cultures of early SE—hypocotyls (HY), nonembryogenic calli (NEC) and primary embryogenic calli (PEC)—were selected to establish the transcriptional profiles. Our data revealed that a total of 62,562 transcripts were present amongst different developmental stages in the two genotypes. Of these, 18,394 and 26,514 differentially expressed genes (DEGs) were identified during callus dedifferentiation (NEC-VS-HY) and embryogenic transdifferentiation (PEC-VS-NEC), respectively in the recalcitrant genotype, 21,842 and 22,343 DEGs in the highly embryogenic genotype. Furthermore, DEGs were clustered into six expression patterns during cotton SE process in the two genotypes. Moreover, functional enrichment analysis revealed that DEGs were significantly enriched in fatty acid, tryptophan and pyruvate metabolism in the highly embryogenic genotype and in DNA conformation change otherwise in the recalcitrant genotype. In addition, critical SE-associated expressed transcription factors, as well as alternative splicing events, were notably and preferentially activated during embryogenic transdifferentiation in the highly embryogenic genotype compared with the recalcitrant genotype. Taken together, by systematically comparing two genotypes with distinct embryogenic abilities, the findings in our study revealed a comprehensive overview of the dynamic gene regulatory patterns and uncharacterized complex regulatory pathways during cotton SE genotype-dependent response. Our work provides insights into the molecular basis and important gene resources for understanding the underlying genotype recalcitrance during SE process and plant regeneration, thereby holding great promise for accelerating the application of biotechnology to cotton for improving its breeding efficiency.

Plant somatic cells can reprogram into differentiated embryos through somatic embryogenesis (SE) on the condition of plant growth regulators (PGRs). RNA sequencing analysis was performed to investigate transcriptional profiling on cotton redifferentiated callus that was induced by different auxin types (IAA and 2,4-D), different concentrations (0, 0.025, and 0.05 mg L⁻¹), and different incubation times (0, 5, and 20 days). Under the 2,4-D induction effect, signal transduction pathways of plant hormones were significantly enriched in the embryogenic response stage (5 days). These results indicated that auxin signal transduction genes were necessary for the initial response of embryogenic differentiation. In the pre-embryonic initial period (20 days), the photosynthetic pathway was significantly enriched. Most differentially expressed genes (DEGs) were downregulated under the induction of 2,4-D. Upon the dose effect of IAA and 2,4-D, respectively, pathways were significantly enriched in phenylpropanoid biosynthesis, fatty acid metabolism, and carbon metabolic pathways. Therefore, primary and secondary metabolism pathways were critical in cotton SE. These results showed that complex synergistic mechanisms involving multiple cellular pathways were the causes of the induction and dose effect of auxin-induced SE. This study reveals a systematic molecular response to auxin signals and reveals the way that regulates embryogenic redifferentiation during cotton SE.

Understanding somatic cell totipotency remains a challenge facing scientific inquiry today. Plants display remarkable cell totipotency expression, illustrated by single-cell differentiation during somatic embryogenesis (SE) for plant regeneration. Determining cell identity and exploring gene regulation in such complex heterogeneous somatic cell differentiation have been major challenges. Here, we performed high-throughput single-cell sequencing assays to define the precise cellular landscape and revealed the modulation mode of marker genes during embryogenic differentiation in cotton (Gossypium hirsutum L.) as the crop for biotechnology application. We demonstrated that nonembryogenic calli (NEC) and primary embryogenic calli (PEC) tissues were composed of heterogeneous cells that could be partitioned into 4 broad populations with 6 distinct cell clusters. Enriched cell clusters and cell states were identified in NEC and PEC samples, respectively. Moreover, a broad repertoire of new cluster-specific genes and associated expression modules were identified. The energy metabolism, signal transduction, environmental adaptation, membrane transport pathways, and a series of transcription factors were preferentially enriched in cell embryogenic totipotency expression. Notably, the SE-ASSOCIATED LIPID TRANSFER PROTEIN (SELTP) gene dose-dependently marked cell types with distinct embryogenic states and exhibited a parabolic curve pattern along the somatic cell embryogenic differentiation trajectory, suggesting that SELTP could serve as a favorable quantitative cellular marker for detecting embryogenic expression at the single-cell level. In addition, RNA velocity and Scissor analysis confirmed the pseudo-temporal model and validated the accuracy of the scRNA-seq data, respectively. This work provides valuable marker-genes resources and defines precise cellular taxonomy and trajectory atlases for somatic cell embryogenic differentiation in plant regeneration.

The somatic embryogenesis (SE) process of plants is regulated by exogenous hormones. During the SE, different genes sensitively respond to hormone signals through complex regulatory networks to exhibit plant totipotency. When cultured in indole-3-butyric acid (IBA) concentration gradient medium supplemented with 0 mg dm⁻³, 0.025 mg dm⁻³, and 0.05 mg dm⁻³ IBA, the callus differentiation rate first increased then decreased in cotton. To characterize the molecular basis of IBA-induced regulating SE, transcriptome analysis was conducted on embryogenic redifferentiation. Upon the examination of the IBA’s embryogenic inductive effect, it was revealed that pathways related to plant hormone signal transduction and alcohol degradation were significantly enriched in the embryogenic responsive stage (5 days). The photosynthesis, alcohol metabolism and cell cycle pathways were specifically regulated in the pre-embryonic initial period (20 days). Upon the effect of the IBA dose, in the embryogenic responsive stage (5 days), the metabolism of xenobiotics by the cytochrome P450 pathway and secondary metabolism pathways of steroid, flavonoid, and anthocyanin biosynthesis were significantly enriched. The phenylpropanoid, brassinosteroid, and anthocyanin biosynthesis pathways were specifically associated in the pre-embryonic initial period (20 days). At different developmental stages of embryogenic induction, photosynthesis, flavonoid biosynthesis, phenylpropanoid biosynthesis, mitogen-activated protein kinase (MAPK) signaling, xenobiotics metabolism by cytochrome P450, and brassinosteroid biosynthesis pathways were enriched at low a IBA concentration. Meanwhile, at high IBA concentration, the carbon metabolism, alcohol degradation, circadian rhythm and biosynthesis of amino acids pathways were significantly enriched. The results reveal that complex regulating pathways participate in the process of IBA-induced redifferentiation in cotton somatic embryogenesis. In addition, collections of potential essential signaling and regulatory genes responsible for dose IBA-induced efficient embryogenic redifferentiation were identified. Quantitative real-time PCR (qRT-PCR) was performed on the candidate genes with different expression patterns, and the results are basically consistent with the RNA-seq data. The results suggest that the complicated and concerted IBA-induced mechanisms involving multiple cellular pathways are responsible for dose-dependent plant growth regulator-induced SE. This report represents a systematic study and provides new insight into molecular signaling and regulatory basis underlying the process of dose IBA-induced embryogenic redifferentiation during SE.