Metabolome profiling of cacao (Theobroma cacao L.) callus under drought stress conditions induced by polyethylene glycol (PEG) as osmoticant.

INTRODUCTION: The cacao tree (Theobroma cacao), a perennial crop that serves as a source of cacao beans, can suffer from drastic climate changes such as irregular rainfall and shorter rainy seasons. The search for hybrids which are capable of producing specific metabolites favoring adaptation in new climatic conditions is a challenge in cacao farming. OBJECTIVES: We aimed to (1) analyze the metabolic changes in calli of three cacao genotypes during water deficit induced by incubation with polyethylene glycol and (2) assess their response to water deficit stress with regard to somatic embryo differentiation. METHODS: Metabolic profiling was carried out using 1 H-NMR spectroscopy and multivariate data analysis was applied to crude extracts of calli grown in non-stress or water deficit stress conditions. RESULTS: Water deficit stress influences the capacity of calli to produce embryos. The SCA12 genotype exhibited the best conversion capacity under severe conditions and was considered as tolerant to drought, followed by the SCA6 genotype (mid-tolerant) and the MA12 genotype (sensitive). Fifty-four metabolites were identified in the three cacao genotypes and discriminant metabolites were identified. Metabolites involved in water stress tolerance such as fructose, trans-aconitic acid, leucine, and hydroxybenzene derivatives were observed in SCA12, the tolerant genotype. CONCLUSION: These results demonstrate the utility of 1 H-NMR metabolomics as an essential tool for the analysis of the drought tolerance characteristics of T. cacao.

Dynamics of Auxin and Cytokinin Metabolism during Early Root and Hypocotyl Growth in Theobroma cacao.

The spatial location and timing of plant developmental events are largely regulated by the well balanced effects of auxin and cytokinin phytohormone interplay. Together with transport, localized metabolism regulates the concentration gradients of their bioactive forms, ultimately eliciting growth responses. In order to explore the dynamics of auxin and cytokinin metabolism during early seedling growth in Theobroma cacao (cacao), we have performed auxin and cytokinin metabolite profiling in hypocotyls and root developmental sections at different times by using ultra-high-performance liquid chromatography-electrospray tandem mass spectrometry (UHPLC-MS/MS). Our work provides quantitative characterization of auxin and cytokinin metabolites throughout early root and hypocotyl development and identifies common and distinctive features of auxin and cytokinin metabolism during cacao seedling development.

Effect of cacao bean quality grade on cacao quality evaluation by cut test and correlations with free amino acids and polyphenols profiles.

Cacao fermentation induces biochemical changes in the beans that lead to different cacao grades such as slaty (unfermented), violet (under fermented) and brown (fully fermented) beans. This grade heterogeneity in a sample set can impact the quality of the end-products. In the present study, amino acids and polyphenol contents of slaty, violet and brown beans were evaluated. Free amino acids were derivatized from o-phthalaldehyde and detected with a fluorescence spectrophotometer coupled with a reverse phase HPLC system. Polyphenols were analysed at 280 nm by HPLC using a Photodiode Array Detector. Amino acids content in the violet beans were significantly higher (11,165 ± 4281 mg kg-1 fat free dry material, ffdm) than that of the slaty beans (4304.5 ± 1927.6 mg kg-1 ffdm), meanwhile there was no significant difference between violet and brown beans. Epicatechin, catechin, cyanidin-3-arabinoside and cyanidin-3-galactoside contents were significantly lower in violet and brown beans when compared to slaty beans. Our results have shown that a mixture of violet and brown beans is suitable to obtain polyphenols and amino acids in the development of cacao products.

Cassava postharvest physiological deterioration: a complex phenomenon involving calcium signaling, reactive oxygen species and programmed cell death.

Postharvest physiological deterioration (PPD) of cassava (Manihot esculenta) storage roots is a complex physiological and biochemical process which involve many regulatory networks linked with specific proteins modulation and signaling transduction pathways. However, it is poorly understood regarding biological regulation, and the interactions among protein groups and signals to determine PPD syndrome in cassava storage roots. This review sheds some light on the possible molecular mechanisms involved in reactive oxygen species (ROS), calcium signaling transduction, and programmed cell death (PCD) in cassava PPD syndrome. A model for predicting crosstalk among calcium signaling, ROS and PCD is suggested to fine-tune PPD syndrome. This would clues to cassava molecular breeding to alleviate the PPD effects on the shelf-life.

Fate of Anthocyanins through Cocoa Fermentation. Emergence of New Polyphenolic Dimers.

Fresh, ripe cocoa beans from Cameroon (German cocoa/Amelonado group and ICS 40/Trinitario group) were subjected to fermentation-like incubations in acetic acid, lactic acid, or both and to natural fermentation. Two naturally fermented samples from Cuba (UF 654/Trinitario group and C 411/Criollo group) were also investigated. Both cyanidin-3-galactoside and cyanidin-3-arabinoside (found as major anthocyanins in colored beans only) were drastically degraded through fermentation, especially in small beans and in the presence of acetic acid. On the other hand, emergence of a cyanidin-rhamnose isomer was evidenced, even in Criollo beans. In addition to the recently described structures F1 and F2 [m/z = 575 in ESI(-)], three additional polyphenolic structures [F3, F4, and F5; m/z = 557 in ESI(+)] were found after fermentation, the two former ones resulting from epicatechin oxidation. Synthesis of F5 requires an interclass reaction between cyani(di)n and epicatechin, which explains its absence in fermented Criollo beans.

Proteome analysis during pod, zygotic and somatic embryo maturation of Theobroma cacao.

Two dimensional electrophoresis and nano-LC-MS were performed in order to identify alterations in protein abundance that correlate with maturation of cacao zygotic and somatic embryos. The cacao pod proteome was also characterized during development. The recently published cacao genome sequence was used to create a predicted proteolytic fragment database. Several hundred protein spots were resolved on each tissue analysis, of which 72 variable spots were subjected to MS analysis, resulting in 49 identifications. The identified proteins represent an array of functional categories, including seed storage, stress response, photosynthesis and translation factors. The seed storage protein was strongly accumulated in cacao zygotic embryos compared to their somatic counterpart. However, sucrose treatment (60 g L(-1)) allows up-regulation of storage protein in SE. A high similarity in the profiles of acidic proteins was observed in mature zygotic and somatic embryos. Differential expression in both tissues was observed in proteins having high pI. Several proteins were detected exclusively in fruit tissues, including a chitinase and a 14-3-3 protein. We also identified a novel cacao protein related to known mabinlin type sweet storage proteins. Moreover, the specific presence of thaumatin-like protein, another sweet protein, was also detected in fruit tissue. We discuss our observed correlations between protein expression profiles, developmental stage and stress responses.

Genome-wide analysis reveals divergent patterns of gene expression during zygotic and somatic embryo maturation of Theobroma cacao L., the chocolate tree.

BACKGROUND: Theobroma cacao L. is a tropical fruit tree, the seeds of which are used to create chocolate. In vitro somatic embryogenesis (SE) of cacao is a propagation system useful for rapid mass-multiplication to accelerate breeding programs and to provide plants directly to farmers. Two major limitations of cacao SE remain: the efficiency of embryo production is highly genotype dependent and the lack of full cotyledon development results in low embryo to plant conversion rates. With the goal to better understand SE development and to improve the efficiency of SE conversion we examined gene expression differences between zygotic and somatic embryos using a whole genome microarray. RESULTS: The expression of 28,752 genes was determined at 4 developmental time points during zygotic embryogenesis (ZE) and 2 time points during cacao somatic embryogenesis (SE). Within the ZE time course, 10,288 differentially expressed genes were enriched for functions related to responses to abiotic and biotic stimulus, metabolic and cellular processes. A comparison ZE and SE expression profiles identified 10,175 differentially expressed genes. Many TF genes, putatively involved in ethylene metabolism and response, were more strongly expressed in SEs as compared to ZEs. Expression levels of genes involved in fatty acid metabolism, flavonoid biosynthesis and seed storage protein genes were also differentially expressed in the two types of embryos. CONCLUSIONS: Large numbers of genes were differentially regulated during various stages of both ZE and SE development in cacao. The relatively higher expression of ethylene and flavonoid related genes during SE suggests that the developing tissues may be experiencing high levels of stress during SE maturation caused by the in vitro environment. The expression of genes involved in the synthesis of auxin, polyunsaturated fatty acids and secondary metabolites was higher in SEs relative to ZEs despite lack of lipid and metabolite accumulation. These differences in gene transcript levels associated with critical processes during seed development are consistent with the fact that somatic embryos do not fully develop the large storage cotyledons found in zygotic embryos. These results provide insight towards design of improved protocols for cacao somatic embryogenesis.

Comparative proteomic analysis of early somatic and zygotic embryogenesis in Theobroma cacao L.

Somatic embryogenesis can efficiently foster the propagation of Theobroma cacao, but the poor quality of resulted plantlet hinders the use of this technique in the commercial scale. The current study has been initiated to systematically compare the physiological mechanisms underlying somatic and zygotic embryogenesis in T. cacao on the proteome level. About 1000 protein spots per fraction could be separated by two-dimensional isoelectric focusing/SDS PAGE. More than 50 of the protein spots clearly differed in abundance between zygotic and somatic embryos: 33 proteins spots were at least 3-fold higher in abundance in zygotic embryos and 20 in somatic embryos. Analyses of these protein spots differing in volume by mass spectrometry resulted in the identification of 68 distinct proteins. Many of the identified proteins are involved in genetic information processing (21 proteins), carbohydrate metabolism (11 proteins) and stress response (7 proteins). Somatic embryos especially displayed many stress related proteins, few enzymes involved in storage compound synthesis and an exceptional high abundance of endopeptidase inhibitors. Phosphoenolpyruvate carboxylase, which was accumulated more than 3-fold higher in zygotic embryos, represents a prominent enzyme in the storage compound metabolism in cacao seeds. Implications on the improvement of somatic embryogenesis in cacao are discussed.

Regeneration of somatic embryos in Theobroma cacao L. in temporary immersion bioreactor and analyses of free amino acids in different tissues.

The present study aimed at developing temporary immersion bioreactor techniques for multiplication of cacao somatic embryos. Temporary Immersion System (TIS), i.e. flooding of plant tissue at regular time intervals provides an efficient way to propagate plants. Somatic embryos were regenerated in twin flask bioreactors. The TIS proved to be suitable for mass regeneration of somatic embryos and for their subsequent direct sowing. The number of embryos after 3 months of culture was significantly higher in TIS cultures than in the solid medium variant. TIS also improved embryo development regarding the conversion to torpedo shaped forms. Matured embryos derived from TIS and pre-treated with 6% sucrose were converted into plants after direct sowing. Additionally to the influence of culture conditions on the development of somatic embryogenesis the content and composition of free amino acids were analysed. The content of free amino acids in somatic embryos rose as immersion frequency increased. The endogenous free GABA content in embryogenic callus was significantly higher than in non-embryogenic callus.

Characterization of leafy cotyledon1-like during embryogenesis in Theobroma cacao L.

Theobroma cacao L., an economically important crop for developing countries, can be experimentally propagated by somatic embryogenesis. Because of their potential roles in embryogenesis, a gene candidate strategy was initiated to find gene homologues of the members of the leafy cotyledon family of transcription factors. A homologue of the leafy cotyledon1-like gene, that encodes the HAP 3 subunit of the CCAAT box-binding factor, was found in the cocoa genome (TcL1L). The translated peptide shared a high amino acid sequence identity with the homologous genes of Arabidopsis thaliana, Phaseolus coccineus and Helianthus annuus. TcL1L transcripts mainly accumulated in young and immature zygotic embryos, and, to a lesser extent, in young and immature somatic embryos. In situ hybridization specified the localization of the transcripts as being mainly in embryonic cells of young embryos, the meristematic cells of the shoot and root apex of immature embryos, and in the protoderm and epidermis of young and immature embryos, either zygotic or somatic. Non-embryogenic explants did not show TcL1L expression. Ectopic expression of the TcL1L gene could partially rescue the Arabidopsis lec1 mutant phenotype, suggesting a similarity of function in zygotic embryogenesis.

Pluripotent versus totipotent plant stem cells: dependence versus autonomy?

Little is known of the mechanisms that induce the dedifferentiation of a single somatic cell into a totipotent embryogenic cell that can either be regenerated or develop into an embryo and subsequently an entire plant. In this Opinion article, we examine the cellular, physiological and molecular similarities and differences between different plant stem cell types. We propose to extend the plant stem cell concept to include single embryogenic cells as a totipotent stem cell based on their capacity to regenerate or develop into an embryo under certain conditions. Our survey suggests that differences in chromatin structure might ensure that meristem-localized stem cells have supervised freedom and are pluripotent, and that embryogenic stem cells are unsupervised, autonomous and, hence, freely totipotent.