Transcriptomic analyses of cacao cell suspensions in light and dark provide target genes for controlled flavonoid production.

Catechins, including catechin (C) and epicatechin (E), are the main type of flavonoids in cacao seeds. They play important roles in plant defense and have been associated with human health benefits. Although flavonoid biosynthesis has been extensively studied using in vitro and in vivo models, the regulatory mechanisms controlling their accumulation under light/dark conditions remain poorly understood. To identify differences in flavonoid biosynthesis (particularly catechins) under different light treatments, we used cacao cell suspensions exposed to white-blue light and darkness during 14 days. RNA-Seq was applied to evaluate differential gene expression. Our results indicate that light can effectively regulate flavonoid profiles, inducing a faster accumulation of phenolic compounds and shifting E/C ratios, in particular as a response to switching from white to blue light. The results demonstrated that HY5, MYB12, ANR and LAR were differentially regulated under light/dark conditions and could be targeted by overexpression aiming to improve catechin synthesis in cell cultures. In conclusion, our RNA-Seq analysis of cacao cells cultured under different light conditions provides a platform to dissect key aspects into the genetic regulatory network of flavonoids. These light-responsive candidate genes can be used further to modulate the flavonoid production in in vitro systems with value-added characteristics.

A rational approach for the improvement of biomass production and lipid profile in cacao cell suspensions.

Cocoa butter (CB) is produced in the seeds of Theobroma cacao representing 50% of its dry weight. The lipid composition plays an important role in the physicochemical, rheological, and sensory properties of the CB, making this fat a valuable resource for the production of chocolates, cosmetics, and pharmaceuticals. In this paper, are described experimental strategies used for a rational improvement of biomass production and fatty acids in cacao cell suspension cultures. First, the lipid profile in four cacao varieties is characterized, and then, one variety is selected to induce cell suspensions using a direct method without previous establishment of a callus phase. To improve growth and total fat production in cell suspension cultures, modified DKW media and newly designed media culture, based on the mineral concentrations of cacao seeds (cacao biomass production, "CBP"), are analyzed and compared. In addition, the effect of acetate in the lipid profile of cell suspensions is evaluated. Ultrastructural histological analysis of lipid vesicles in cacao seeds and cell suspensions is also performed. The results will show that it is feasible to establish cacao suspensions without the calli step and increase the biomass production by selecting a suitable cacao variety and tissue and also applying a new culture media formulation. In addition, it is possible to synthesize fatty acids in cell cultures and modify the lipid profile adding a precursor of the novo biosynthesis of fatty acids such as the acetate. Transmission electronic microscopy examinations and differential interference contrast microscopy analysis will demonstrate that lipid vesicles are the main reserve substance in both cacao seeds and cell suspensions.

Biochemical precursor effects on the fatty acid production in cell suspension cultures of Theobroma cacao L.

Cocoa butter (CB) is composed of 96% palmitic, stearic, oleic, linoleic and linolenic fatty acids that are responsible for the hardness, texture and fusion properties of chocolate. Through in vitro plant cell culture it is possible to modify CB lipid profiles and to study the fatty acid biosynthesis pathway on a subcellular level, evaluating fundamental aspects to enhance in vitro fatty acid production in a specific and controlled way. In this research, culture media was supplemented with acetate, biotin, pyruvate, bicarbonate and glycerol at three different concentrations and the effects on the biomass production (g/L), cell viability, and fatty acids profile and production was evaluated in in vitro cell suspensions culture. It was found that biotin stimulated fatty acid synthesis without altering cell viability and cell growth. It was also evident a change in the lipid profile of cell suspensions, increasing middle and long chain fatty acids proportion, which are unusual to those reported in seeds; thus implying that it is possible to modify lipid profiles according to the treatment used. According to the results of sucrose gradients and enzyme assays performed, it is proposed that cacao cells probably use the pentose phosphate pathway, mitochondria being the key organelle in the carbon flux for the synthesis of reductant power and fatty acid precursors.

Effect of Cryopreservation and Post-Cryopreservation Somatic Embryogenesis on the Epigenetic Fidelity of Cocoa (Theobroma cacao L.).

UNLABELLED: While cocoa plants regenerated from cryopreserved somatic embryos can demonstrate high levels of phenotypic variability, little is known about the sources of the observed variability. Previous studies have shown that the encapsulation-dehydration cryopreservation methodology imposes no significant extra mutational load since embryos carrying high levels of genetic variability are selected against during protracted culture. Also, the use of secondary rather than primary somatic embryos has been shown to further reduce the incidence of genetic somaclonal variation. Here, the effect of in vitro conservation, cryopreservation and post-cryopreservation generation of somatic embryos on the appearance of epigenetic somaclonal variation were comparatively assessed. To achieve this we compared the epigenetic profiles, generated using Methylation Sensitive Amplified Polymorphisms, of leaves collected from the ortet tree and from cocoa somatic embryos derived from three in vitro conditions: somatic embryos, somatic embryos cryopreserved in liquid nitrogen and somatic embryos generated from cryoproserved somatic embryos. Somatic embryos accumulated epigenetic changes but these were less extensive than in those regenerated after storage in LN. Furthermore, the passage of cryopreserved embryos through another embryogenic stage led to further increase in variation. Interestingly, this detected variability appears to be in some measure reversible. The outcome of this study indicates that the cryopreservation induced phenotypic variability could be, at least partially, due to DNA methylation changes. KEY MESSAGE: Phenotypic variability observed in cryostored cocoa somatic-embryos is epigenetic in nature. This variability is partially reversible, not stochastic in nature but a directed response to the in-vitro culture and cryopreservation.

Apoplastic and intracellular plant sugars regulate developmental transitions in witches' broom disease of cacao.

Witches' broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant-fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation.

High-resolution transcript profiling of the atypical biotrophic interaction between Theobroma cacao and the fungal pathogen Moniliophthora perniciosa.

Witches' broom disease (WBD), caused by the hemibiotrophic fungus Moniliophthora perniciosa, is one of the most devastating diseases of Theobroma cacao, the chocolate tree. In contrast to other hemibiotrophic interactions, the WBD biotrophic stage lasts for months and is responsible for the most distinctive symptoms of the disease, which comprise drastic morphological changes in the infected shoots. Here, we used the dual RNA-seq approach to simultaneously assess the transcriptomes of cacao and M. perniciosa during their peculiar biotrophic interaction. Infection with M. perniciosa triggers massive metabolic reprogramming in the diseased tissues. Although apparently vigorous, the infected shoots are energetically expensive structures characterized by the induction of ineffective defense responses and by a clear carbon deprivation signature. Remarkably, the infection culminates in the establishment of a senescence process in the host, which signals the end of the WBD biotrophic stage. We analyzed the pathogen's transcriptome in unprecedented detail and thereby characterized the fungal nutritional and infection strategies during WBD and identified putative virulence effectors. Interestingly, M. perniciosa biotrophic mycelia develop as long-term parasites that orchestrate changes in plant metabolism to increase the availability of soluble nutrients before plant death. Collectively, our results provide unique insight into an intriguing tropical disease and advance our understanding of the development of (hemi)biotrophic plant-pathogen interactions.

Altered physiology, cell structure, and gene expression of Theobroma cacao seedlings subjected to Cu toxicity.

Seedlings of Theobroma cacao CCN 51 genotype were grown under greenhouse conditions and exposed to increasing concentrations of Cu (0.005, 1, 2, 4, 8, 16, and 32 mg Cu L(-1)) in nutrient solution. When doses were equal or higher than 8 mg Cu L(-1), after 24 h of treatment application, leaf gas exchange was highly affected and changes in chloroplasts thylakoids of leaf mesophyll cells and plasmolysis of cells from the root cortical region were observed. In addition, cell membranes of roots and leaves were damaged. In leaves, 96 h after treatments started, increases in the percentage of electrolyte leakage through membranes were observed with increases of Cu in the nutrient solution. Moreover, there was an increase in the concentration of thiobarbituric acid-reactive substances in roots due to lipid peroxidation of membranes. Chemical analysis showed that increases in Cu concentrations in vegetative organs of T. cacao increased with the increase of the metal in the nutrient solution, but there was a greater accumulation of Cu in roots than in shoots. The excess of Cu interfered in the levels of Mn, Zn, Fe, Mg, K, and Ca in different organs of T. cacao. Analysis of gene expression via RTq-PCR showed increased levels of MT2b, SODCyt, and PER-1 expression in roots and of MT2b, PSBA, PSBO, SODCyt, and SODChI in leaves. Hence, it was concluded that Cu in nutrient solution at doses equal or above 8 mg L(-1) significantly affected leaf gas exchange, cell ultrastructure, and transport of mineral nutrients in seedlings of this T. cacao genotype.

Karyotype variation in cultivars and spontaneous cocoa mutants (Theobroma cacao L.).

Four mutant cocoa accessions with morphological changes and a cultivar sample were karyomorphologically characterized. Slides were prepared by enzymatic digestion of the root meristem and squashed in 45% acetic acid, followed by 2% Giemsa staining. The chromosome number of 2n = 20 was seen in all accessions. The karyotype formula for Cacau Comum and Cacau Rui was 2n = 20m. Submetacentric chromosomes were observed in Cacau Pucala and Cacau Jaca, both with 2n = 18m + 2sm, but the karyotype formula for Cacau Sem Vidro was 2n = 16m + 4sm. Satellites were located on the long arm of the 1st and 2nd chromosome pairs of Cacau Comum, whereas Cacau Pucala had satellites on the 6th chromosome pair. Greater karyotypic variation in Cacau Sem Vidro was found, whose 1st and 2nd chromosome pairs had satellites on the long arm and 6th and 10th pairs had satellites on the short arm. Analysis revealed a lower average chromosome length in Cacau Comum (1.53 ± 0.026 µm) and a higher length in Cacau Sem Vidro (2.26 ± 0.038 µm). ANOVA revealed significant difference (P < 0.01) for the average chromosome length and the length of chromosome pairs within and between accessions. The average chromosome lengths of mutants of Cacau Rui and Cacau Jaca were not statistically different by the Tukey test at 5% probability. The karyotypic diversity observed in this study is not necessarily associated with the changing character of the accessions analyzed, but may reflect the genetic variation observed in Theobroma cacao.

Confocal observations of late-acting self-incompatibility in Theobroma cacao L.

Cocoa (Theobroma cacao) has an idiosyncratic form of late-acting self-incompatibility that operates through the non-fusion of incompatible gametes. Here, we used high-resolution confocal microscopy to define fine level changes to the embryo sac of the strongly self-incompatible cocoa genotype SCA 24 in the absence of pollination, and following compatible and incompatible pollination. All sperm nuclei had fused with the female nuclei by 48 h following compatible pollinations. However, following incompatible pollinations, we observed divergence in the behaviour of sperm nuclei following release into the embryo sac. Incomplete sperm nucleus migration occurred in approximately half of the embryo sacs, where the sperm nuclei had so far failed to reach the female gamete nuclei. Sperm nuclei reached but did not fuse with the female gamete nuclei in the residual cases. We argue that the cellular mechanisms governing sperm nucleus migration to the egg nucleus and those controlling subsequent nuclear fusion are likely to differ and should be considered independently. Accordingly, we recommend that future efforts to characterise the genetic basis of LSI in cocoa should take care to differentiate between these two events, both of which contribute to failed karyogamy. Implications of these results for continuing efforts to gain better understanding of the genetic control of LSI in cocoa are discussed.

Carbon source-induced changes in the physiology of the cacao pathogen Moniliophthora perniciosa (Basidiomycetes) affect mycelial morphology and secretion of necrosis-inducing proteins.

Quantitative and qualitative relationships were found between secreted proteins and their activity, and the hyphal morphology of Moniliophthora perniciosa, the causal agent of witches' broom disease in Theobroma cacao. This fungus was grown on fermentable and non-fermentable carbon sources; significant differences in mycelial morphology were observed and correlated with the carbon source. A biological assay performed with Nicotiana tabacum leaves revealed that the necrosis-related activity of extracellular fungal proteins also differed with carbon source. There were clear differences in the type and quantity of the secreted proteins. In addition, the expression of the cacao molecular chaperone BiP increased after treatment with secreted proteins, suggesting a physiological response to the fungus secretome. We suggest that the carbon source-dependent energy metabolism of M. perniciosa results in physiological alterations in protein expression and secretion; these may affect not only M. perniciosa growth, but also its ability to express pathogenicity proteins.

Detection and quantification of in vitro-culture induced chimerism using simple sequence repeat (SSR) analysis in Theobroma cacao (L.).

Mutation rates are often elevated in plants regenerated from in vitro culture, giving rise to so-called 'somaclonal variation'. Detailed characterisation of mutation profiles that arise during culture should improve our understanding of processes influencing mutation and allow the selection of protocols yielding the fewest/least severe changes. Somatic mutations will usually produce genetic chimeras where unchanged alleles are retained by some cells. Such chimeras are difficult to detect but likely to form a significant proportion of any regenerant population. We present a simple protocol that enables the provisional diagnosis of both homogenous and chimeric mutants among large regenerant populations, together with a semi-quantitative means of estimating the proportion of mutant cells. The assay exploits consistent differential amplification of alternate simple sequence repeat alleles at heterozygous loci. Calibration of the relative amplification of alleles from two genotypes-and the synthetic chimeras created from them-revealed a strong linear relationship between 'peak heights' representing alternate alleles following capillary electrophoresis. The assay predicts chimeric composition to a reasonable level of confidence (+/-5%) so long as the infrequent allele exceeds 15% of the template. The system was applied to 233 regenerants of cocoa somatic embryogenesis and identified 72 (31%) putative chimeric mutants for slippage mutation or allele loss across two loci.

Changes in nucleus, nucleolus and cell size accompanying somatic embryogenesis of Theobroma cacao L. I. Relationship between DNA and total protein content and size of nucleus, nucleolus and cell.

There was a linear relation between an increase in DNA content and size of nuclei, nucleoli and cells in callus and proembryos (Theobroma cacao L.). In callus the increase of DNA content was accompanied by proportional increase in nuclear size whereas in proembryos the increase in nuclear size did not match the increasing amount of DNA. The stimulation of embryogenesis by 10(-2) mg/l 2,4-D was associated with increase in nuclear and nucleolar size and with decrease in cell sizes. Inhibition of embryogenesis by 1.0 mg/l 2,4-D+10% coconut water did not change nuclear size, but increased cell size in relation to the control. The process of embryo formation was accompanied by changes in relationship between nuclear, nucleolar and cell size and the total (DNFB-stained) proteins content. In callus as well as in proembryo the increase in total protein content in nucleus was not equivalent to the increasing sizes of nuclei which leads to the decrease in nuclear protein concentration. Similar situation was observed for nucleoli. Differences were found in the concentration of cytoplasmic proteins between the callus and proembryo cells. The stimulation of embryogenesis by low concentration of 2,4-D resulted in decrease in concentration of total proteins in nuclei and nucleoli and the increase in cytoplasm.

Changes in nucleus, nucleolus and cell size accompanying somatic embryogenesis of Theobroma cacao L. II. Relation between basic protein content and size of nucleus, nucleolus and cell.

Embryo formation from callus of Theobroma cacao L. was associated with the changes in relationship between nuclear, nucleolar and cell sizes and the content of basic proteins (FG-FCF-stained). Together with the increase in nuclear size of callus and proembryo cells the increase in the amount of nuclear basic proteins was found. In the callus cells the increase in nucleolar protein content exceeded that in nucleolus size, which led to the rise in basic protein concentration in the nucleolus. However, in the early stage of embryogenesis the increase in protein content was not so marked as that in callus, which indicated that embryogenesis involved a decrease in concentration of nucleolar basic proteins. Differences between callus and proembryo cells were also observed in the concentration of cytoplasmic proteins. The increase in size of callus cells was the same as the increasing amount of cytoplasmic proteins. In proembryos a significant increase in cell size was accompanied by only slight changes in cytoplasmic proteins. The stimulation of embryogenesis by 2,4-D resulted in an increase of nuclear concentration of basic proteins in proembryos. The intensification of embryogenesis involved the decrease of the concentration of nucleolar proteins together with the increase in concentration of basic cytoplasmic proteins.