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Unravelling the Metabolic and Hormonal Machinery During Key Steps of Somatic Embryogenesis: A Case Study in Coffee.

ABSTRACT: Somatic embryogenesis (SE) is one of the most promising processes for large-scale dissemination of elite varieties. However, for many plant species, optimizing SE protocols still relies on a trial-and-error approach. Using coffee as a model plant, we report here the first global analysis of metabolome and hormone dynamics aiming to unravel mechanisms regulating cell fate and totipotency. Sampling from leaf explant dedifferentiation until embryo development covered 15 key stages. An in-depth statistical analysis performed on 104 metabolites revealed that massive re-configuration of metabolic pathways induced SE. During initial dedifferentiation, a sharp decrease in phenolic compounds and caffeine levels was also observed while auxins, cytokinins and ethylene levels were at their highest. Totipotency reached its highest expression during the callus stages when a shut-off in hormonal and metabolic pathways related to sugar and energetic substance hydrolysis was evidenced. Abscisic acid, leucine, maltotriose, myo-inositol, proline, tricarboxylic acid cycle metabolites and zeatin appeared as key metabolic markers of the embryogenic capacity. Combining metabolomics with multiphoton microscopy led to the identification of chlorogenic acids as markers of embryo redifferentiation. The present analysis shows that metabolite fingerprints are signatures of cell fate and represent a starting point for optimizing SE protocols in a rational way.

SUBMITTER: Awada R

PROVIDER: S-EPMC6802359 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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Unravelling the Metabolic and Hormonal Machinery During Key Steps of Somatic Embryogenesis: A Case Study in Coffee.

Awada Rayan R Campa Claudine C Gibault Estelle E Déchamp Eveline E Georget Frédéric F Lepelley Maud M Abdallah Cécile C Erban Alexander A Martinez-Seidel Federico F Kopka Joachim J Legendre Laurent L Léran Sophie S Conéjéro Geneviève G Verdeil Jean-Luc JL Crouzillat Dominique D Breton David D Bertrand Benoît B Etienne Hervé H

International journal of molecular sciences 20190920 19

Somatic embryogenesis (SE) is one of the most promising processes for large-scale dissemination of elite varieties. However, for many plant species, optimizing SE protocols still relies on a trial-and-error approach. Using coffee as a model plant, we report here the first global analysis of metabolome and hormone dynamics aiming to unravel mechanisms regulating cell fate and totipotency. Sampling from leaf explant dedifferentiation until embryo development covered 15 key stages. An in-depth stat ...[more]

PMID: 31547069

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Project description:BackgroundSomatic embryogenesis (SE) is one of the most promising processes for large-scale dissemination of elite varieties. However, for many plant species, optimizing SE protocols still relies on a trial and error approach. We report the first global scale transcriptome profiling performed at all developmental stages of SE in coffee to unravel the mechanisms that regulate cell fate and totipotency.ResultsRNA-seq of 48 samples (12 developmental stages × 4 biological replicates) generated 90 million high quality reads per sample, approximately 74% of which were uniquely mapped to the Arabica genome. First, the statistical analysis of transcript data clearly grouped SE developmental stages into seven important phases (Leaf, Dedifferentiation, Primary callus, Embryogenic callus, Embryogenic cell clusters, Redifferentiation and Embryo) enabling the identification of six key developmental phase switches, which are strategic for the overall biological efficiency of embryo regeneration. Differential gene expression and functional analysis showed that genes encoding transcription factors, stress-related genes, metabolism-related genes and hormone signaling-related genes were significantly enriched. Second, the standard environmental drivers used to control SE, i.e. light, growth regulators and cell density, were clearly perceived at the molecular level at different developmental stages. Third, expression profiles of auxin-related genes, transcription factor-related genes and secondary metabolism-related genes were analyzed during SE. Gene co-expression networks were also inferred. Auxin-related genes were upregulated during dedifferentiation and redifferentiation while transcription factor-related genes were switched on from the embryogenic callus and onward. Secondary metabolism-related genes were switched off during dedifferentiation and switched back on at the onset of redifferentiation. Secondary metabolites and endogenous IAA content were tightly linked with their respective gene expression. Lastly, comparing Arabica embryogenic and non-embryogenic cell transcriptomes enabled the identification of biological processes involved in the acquisition of embryogenic capacity.ConclusionsThe present analysis showed that transcript fingerprints are discriminating signatures of cell fate and are under the direct influence of environmental drivers. A total of 23 molecular candidates were successfully identified overall the 12 developmental stages and can be tested in many plant species to optimize SE protocols in a rational way.

Project description:In somatic embryogenesis, embryos are formed from a single or small group of somatic cells in response to stress and hormonal stimuli. In this study, a high-throughput phosphoproteomic analysis was used to identify and quantify phosphoproteins related to the acquisition of embryogenic competence in sugarcane. Embryogenic (EC) and nonembryogenic (NEC) calli were compared at the multiplication phase, resulting in the identification of 1279 phosphoproteins. After the differential accumulation analysis, 163 phosphoproteins were identified as unique to EC, and nine were unique to NEC, while 51 were upaccumulated, and 40 were downaccumulated in EC compared to NEC. These phosphopeptides were mainly single phosphorylated peptides, with serine residues representing the major site of phosphorylation. The motif-x analysis revealed the enrichment of [xxxpSPxxx], [RxxpSxxx] and [xxxpSDxxx] motifs, which are predicted phosphorylation sites for several kinases related to stress responses. Gene ontology enrichment revealed an overrepresentation of biological processes involved in embryonic development, showing that EC-related phosphoproteins (unique to EC and upaccumulated) were associated with stress responses, regulation of gene expression and epigenetic modifications. The NEC-related phosphoproteins (unique to NEC and downaccumulated) were associated with the translation process. In this sense, EC-related phosphoproteins described as potential regulators of abiotic stress tolerance in response to ABA-induced signals such as OSK3 (a catalytic subunit of SnRK1), ABF1, LEAs, REM4.1 and RD29Bs were identified. On the other hand, the NEC-related phosphoproteins EDR1 and PP2Ac-2 were negative regulators of ABA, suggesting a role of ABA in the acquisition of embryogenic competence linked to stress tolerance. Moreover, EC-related phosphoproteins associated with epigenetic modifications, such as HDA6, HDA19, and TPL, as well as those involved in embryo development, including ASIL1, M3KE1, MCM2, and GRV2, were identified as putative potential regulators of embryogenic competence. Our results provide novel data that may help elucidate the phosphorylation mechanisms controlling the activity, affinity and localization of specific proteins during somatic embryogenesis and suggest a role for these proteins in the hormonal signalling cascade leading to stress adaptation by reprogramming genetic expression.

Project description:BackgroundDarjeeling tea is a globally renowned beverage, which faces numerous obstacles in sexual reproduction, such as self-incompatibility, poor seed germination, and viability, as well as issues with vegetative propagation. Somatic embryogenesis (SE) is a valuable method for rapid clonal propagation of Darjeeling tea. However, the metabolic regulatory mechanisms underlying SE in Darjeeling tea remain largely unknown. To address this, we conducted an integrated metabolomics and transcriptomics analysis of embryogenic callus (EC), globular embryo (GE), and heart-shaped embryo (HE).ResultsThe integrated analyses showed that various genes and metabolites involved in the phenylpropanoid pathway, auxin biosynthesis pathway, gibberellin, brassinosteroid and amino acids biosynthesis pathways were differentially enriched in EC, GE, and HE. Our results revealed that despite highly up-regulated auxin biosynthesis genes YUC1, TAR1 and AAO1 in EC, endogenous indole-3-acetic acid (IAA) was significantly lower in EC than GE and HE. However, bioactive Gibberellin A4 displayed higher accumulation in EC. We also found higher BABY BOOM (BBM) and Leafy cotyledon1 (LEC1) gene expression in GE along with high accumulation of castasterone, a brassinosteroid. Total flavonoids and phenolics levels were elevated in GE and HE compared to EC, especially the phenolic compound chlorogenic acid was highly accumulated in GE.ConclusionsIntegrated metabolome and transcriptome analysis revealed enriched metabolic pathways, including auxin biosynthesis and signal transduction, brassinosteroid, gibberellin, phenylpropanoid biosynthesis, amino acids metabolism, and transcription factors (TFs) during SE in Darjeeling tea. Notably, EC displayed lower endogenous IAA levels, conducive to maintaining differentiation, while higher IAA concentration in GE and HE was crucial for preserving embryo identity. Additionally, a negative correlation between bioactive gibberellin A4 (GA4) and IAA was observed, impacting callus growth in EC. The high accumulation of chlorogenic acid, a phenolic compound, might contribute to the low success rate in GE and HE formation in Darjeeling tea. TFs such as BBM1, LEC1, FUS3, LEA, WOX3, and WOX11 appeared to regulate gene expression, influencing SE in Darjeeling tea.

Project description:Background and objectiveObesity is intricately linked with metabolic disturbances. The comprehensive exploration of metabolomes is important in unravelling the complexities of obesity development. This study was aimed to discern unique metabolite signatures in obese and lean individuals using liquid chromatography-mass spectrometry quadruple time-of-flight (LC-MS/Q-TOF), with the goal of elucidating their roles in obesity.MethodsA total of 160 serum samples (Discovery, n = 60 and Validation, n = 100) of obese and lean individuals with stable Body Mass Index (BMI) values were retrieved from The Malaysian Cohort biobank. Metabolic profiles were obtained using LC-MS/Q-TOF in dual-polarity mode. Metabolites were identified using a molecular feature and chemical formula algorithm, followed by a differential analysis using MetaboAnalyst 5.0. Validation of potential metabolites was conducted by assessing their presence through collision-induced dissociation (CID) using a targeted tandem MS approach.ResultsA total of 85 significantly differentially expressed metabolites (p-value <0.05; -1.5 < FC > 1.5) were identified between the lean and the obese individuals, with the lipid class being the most prominent. A stepwise logistic regression revealed three metabolites associated with increased risk of obesity (14-methylheptadecanoic acid, 4'-apo-beta,psi-caroten-4'al and 6E,9E-octadecadienoic acid), and three with lower risk of obesity (19:0(11Me), 7,8-Dihydro-3b,6a-dihydroxy-alpha-ionol 9-[apiosyl-(1->6)-glucoside] and 4Z-Decenyl acetate). The model exhibited outstanding performance with an AUC value of 0.95. The predictive model underwent evaluation across four machine learning algorithms consistently demonstrated the highest predictive accuracy of 0.821, aligning with the findings from the classical logistic regression statistical model. Notably, the presence of 4'-apo-beta,psi-caroten-4'-al showed a statistically significant difference between the lean and obese individuals among the metabolites included in the model.ConclusionsOur findings highlight the significance of lipids in obesity-related metabolic alterations, providing insights into the pathophysiological mechanisms contributing to obesity. This underscores their potential as biomarkers for metabolic dysregulation associated with obesity.

Dataset Information

Unravelling the Metabolic and Hormonal Machinery During Key Steps of Somatic Embryogenesis: A Case Study in Coffee.

Publications

Unravelling the Metabolic and Hormonal Machinery During Key Steps of Somatic Embryogenesis: A Case Study in Coffee.

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