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PubMed2005

Developmental expression of stress response genes in Theobroma cacao leaves and their response to Nep1 treatment and a compatible infection by Phytophthora megakarya.

Developmental expression of stress response genes in Theobroma cacao leaves and their response to Nep1 and a compatible infection by Phytophthora megakarya were studied. Ten genes were selected to represent genes involved in defense (TcCaf-1, TcGlu1,3, TcChiB, TcCou-1, and TcPer-1), gene regulation (TcWRKY-1 and TcORFX-1), cell wall development (TcCou-1, TcPer-1, and TcGlu-1), or energy production (TcLhca-1 and TcrbcS). Leaf development was separated into unexpanded (UE), young red (YR), immature green (IG), and mature green (MG). Our data indicates that the constitutive defense mechanisms used by cacao leaves differ between different developmental stages. TcWRKY-1 and TcChiB were highly expressed in MG leaves, and TcPer-1, TcGlu-1, and TcCou-1 were highly expressed in YR leaves. TcGlu1,3 was highly expressed in UE and YR leaves, TcCaf-1 was highly expressed in UE leaves, and TcLhca-1 and TcrbcS were highly expressed in IG and MG leaves. NEP1 encodes the necrosis inducing protein Nep1 produced by Fusarium oxysporum and has orthologs in Phytophthora species. Nep1 caused cellular necrosis on MG leaves and young pods within 24 h of application. Necrosis was observed on YR leaves 10 days after treatment. Expression of TcWRKY-1, TcORFX-1, TcPer-1, and TcGlu-1 was enhanced and TcLhca-1 and TcrbcS were repressed in MG leaves after Nep1 treatment. Expression of TcWRKY-1 and TcORFX-1 was enhanced in YR leaves after Nep1 treatment. Infection of MG leaf disks by P. megakarya zoospores enhanced expression of TcGlu-1, TcWRKY-1, and TcPer-1 and repressed expression of TcChiB, TcLhca-1 and TcrbcS. Five of the six genes that were responsive to Nep1 were responsive to infection by P. megakarya. Susceptibility of T. cacao to P. megakarya includes altered plant gene expression and phytotoxic molecules like Nep1 may contribute to susceptibility.

Algal Proteins↗
PubMed1999

[Antibacterial effects of cacao mass on enterohemorrhagic Escherichia coli O157:H7].

The antimicrobial activities of aqueous cacao mass extract against enterohemorrhagic Escherichia coli (EHEC) O157:H7 006 strain were studied. Hot water extract of cacao mass (cocoa extract) was shown to inhibit the growth of EHEC O157:H7 006 strain in PBS or CAYE medium. In addition, the production of verotoxins (types 1 and 2) of EHEC O157:H7 006 strain was significantly inhibited by 8.0% cocoa extract. The cocoa extract did not neutralize the cytotoxity of verotoxins, but had inhibitory effect on adhesion of verotoxins to the target Vero cells. These results demonstrate that cacao mass has antimicrobial effects on EHEC O157:H7.

Bacterial Toxins↗
PubMed1984

Cacao bean shell poisoning in a dog.

Cacao bean shells contain potentially toxic quantities of theobromine, a xanthine compound similar in effects to caffeine and theophylline. A dog, which ingested a lethal quantity of garden mulch made from cacao bean shells, developed severe convulsions and died 17 hours later. Analysis of the stomach contents and the ingested cacao bean shells revealed the presence of lethal amounts of theobromine.

Animals↗
PubMed1983

[Effect of the harvest season on the composition of raw and fermented cotyledons of 2 varieties of cacao and shell fractions].

A study was carried out wherein during the period August 1979 to January 1980, samples of raw and fermented cacao were analyzed monthly. These included two varieties: Arriba, taken from a farm in "Quevedo", and the EET-19, grown in "Pichilingüe" by the Instituto Nacional de Investigaciones Agropecuarias (INIAP). Taking the ear of cacao as a basis, the weight of its main parts was determined. The proximal composition was established in the cotyledons, with significant statistical differences in regard to moisture, protein, and ether extract content according to the month of harvest. As to the fermentation process, differences in moisture, ether extract and ash content were detected; differences in the ether extract and ash content were found between the two varieties. The fat extracted from the cotyledons presented different iodine, saponification and acidity index values between the raw and fermented samples, but none were determined between the varieties; as far as the month of harvest is concerned, differences in the acidity index were observed. The percentage composition of the main fatty acids is reported (palmitic, stearic, oleic, and linoleic acids). In order to suggest possible industrial ways of utilizing the cacao shell by-product which is discarded by the shelling machine, the chemical characteristics of five fractions were determined based on the functioning of the shelling machine. The moisture, protein, ether extract, ash, crude fiber, theobromine, and caffeine contents varied among the fractions, and it was dependent on the broken "nibs" content. Differences in the protein, ether extract, and ash content, according to the months of production, were found. Obviously, the high fat content in fractions A (fine dust) and B (fine ground), which varied from 30 to 11 g/100 g, merits its extraction; the remainder meal has a valuable protein and alkaloid content. The chemical characteristics of the fat extracted from the shell of two fractions were similar to the fat extracted from the cotyledons.

Cacao↗
PubMed1990

Energy costs of flowering, fruiting, and cherelle wilt in cacao.

Groups of 14-year-old cacao (Theobroma cacao L.) trees were hand pollinated, pollinated naturally or had their fruits continuously removed. Wilted and non-wilted cherelles (small fruits) were counted every 2 weeks, fallen flowers were counted weekly and mature pods were harvested monthly from 1983 through 1986. Carbohydrate, lipid and protein contents of wilted cherelles, mature pods and flowers were determined. Continuous removal of fruits caused a constant initiation of flowers and a significant increase in flowering intensity compared with the other treatments. The quantity of cherelles in the hand-pollinated trees was significantly greater than in the naturally pollinated trees. However, the number of mature fruits in both treatments was not statistically different because of increased cherelle wilting in the hand-pollinated trees. The continuously depodded trees allocated approximately 8 and 5 times more assimilate to flower production than the hand- and naturally pollinated trees, respectively. The naturally pollinated trees used only about one third as much energy for the production of flowers and wilted cherelles as the hand-pollinated trees, but about 1.4 times more energy than the depodded trees. It is concluded that fruit set in cacao is regulated by assimilate production and that cherelle wilting is the mechanism whereby the tree adjusts production.

Journal Article↗
PubMedDate not supplied

Simultaneous determination of (+)-catechin and (-)-epicatechin in cacao and its products by high-performance liquid chromatography with electrochemical detection.

A fast single-step extraction procedure and HPLC with electrochemical detection (ECD) in the oxidative mode has been used for the simultaneous, quantitative determination of (+)-catechin (CA) and (-)-epicatechin (EC) in cacao and its products. A UV detector measuring at 280 nm was also used in-line for comparison purposes. The samples were extracted with methanol and applied directly to the reversed-phase column of the HPLC. The high sensitivity of the ECD allowed operation at a low voltage (+0.5 V), but with a high sensitivity for the readily oxidizable CA and EC, thus reducing baseline noise. As a result, ECD had a better sensitivity and resolution than UV detection for the simultaneous quantitative determination of CA and EC in cacao and its products.

Cacao↗
PubMed2004

Isolation, characterization, and development of WRKY genes as useful genetic markers in Theobroma cacao.

There is currently an international effort in improving disease resistance and crop yield in Theobroma cacao L., an economically important crop of the tropics, using marker-assisted selection for breeding. We are developing molecular genetic markers focusing upon gene families involved with disease resistance. One such family is the WRKY proteins, which are plant-specific transcriptional factors associated with regulating defense responses to both abiotic and biotic stresses. Degenerate PCR primers were designed to the highly conserved DNA-binding domain and other conserved motifs of group I and group II, subgroups a-c, WRKY genes. Sixteen individual WRKY fragments were isolated from a mixture of T. cacao DNA using one pair of primers. Of the 16 WRKY loci investigated, seven contained single nucleotide polymorphisms within the intron as detected by sequence comparison of the PCR products. Four of these were successfully converted into molecular markers and mapped in an F2 population by capillary electrophoresis-single strand conformation polymorphism analysis. This is the first report of a pair of degenerate primers amplifying WRKY loci directly from genomic DNA and demonstrates a simple method for developing useful genetic markers from members of a large gene family.

Base Sequence↗
PubMed2003

Stable transformation of Theobroma cacao L. and influence of matrix attachment regions on GFP expression.

We describe a protocol for Agrobacterium-mediated genetic transformation of Theobroma cacao L. using cotyledonary explants from primary somatic embryos (SEs) and A. tumefaciens strain AGL1. Transgenic plants carrying the visible marker, gene green fluorescent protein ( EGFP), the selectable marker gene neomycin phosphotransferase II ( NPTII), the class I chitinase gene from cacao ( Chi), and tobacco nuclear matrix attachment regions (MARs) in different combinations were successfully produced via regeneration of secondary SEs. The presence of the Chi gene or MARs did not influence the number of transgenic plants produced compared to the marker genes alone. However, the inclusion of MARs contributed to increased mean GFP expression in the population of transgenics. Additionally, the presence of MARs reduced the occurrence of gene silencing and stabilized high levels of GFP expression in lines of transgenic plants multiplied via reiterative somatic embryogenesis. Ninety-four transgenic plants were acclimated in a greenhouse and grown to maturity. Detailed growth analysis indicated that there were no differences in various growth parameters between transgenic and non-transgenic SE-derived plants. Seeds produced from two genetic crosses with one of the transgenic lines were analyzed for EGFP expression-a near-perfect 1:1 segregation was observed, indicating that this line resulted from the insertion of a single locus of T-DNA.

Base Sequence↗
PubMed2004

Molecular variability analysis of five new complete cacao swollen shoot virus genomic sequences.

Cacao swollen shoot virus (CSSV), a member of the family Caulimovi-ridae, genus Badnavirus occurs in all the main cacao-growing areas of West Africa. We amplified, cloned and sequenced complete genomes of five new isolates, two originating from Togo and three originating from Ghana. The genome of these five newly sequenced isolates all contain the five putative open reading frames I, II, III, X and Y described for the first sequenced CSSV isolate, Agou1 originating from Togo. Their genomes have been aligned with the genome of Agou1. The nucleotide and amino acid sequence identities between isolates have been calculated and a phylogenetic analysis has been made including other pararetroviruses. Maximum nucleotide sequence variability between complete genomes of CSSV isolates was 29.4%. Geographical differentiation between isolates appears more important than differentiation between mild and severe isolates. ORF X differs greatly in size and sequence between the Togolese isolates Nyongbo2 and Agou1, and the four other isolates, its functional role is therefore clearly questionable.

Badnavirus↗
PubMed2003

Out of the Amazon: Theobroma cacao enters the genomic era.

Theobroma cacao has long been a crop of near-mystical human importance among indigenous Mesoamerican cultures and its importance in modern culture is growing with new realizations of the potential health benefits of cocoa polyphenolic compounds. However, cultivated T. cacao is vulnerable to emerging disease pressures because it has a narrow genetic base, and systematic genetic improvement of the crop is imperative. A wide range of genomic tools and resources has been developed and are providing the basis for genome-based breeding and gene discovery.

Brazil↗
PubMed2000

Cariostatic activity of cacao mass extract.

Chocolate is suspected to contain some caries-inhibitory substances. The cariostatic activity of cacao mass extract (CM), the main component of chocolate, was examined in vitro and in experimental animals. CM showed no detectable effects on the cellular growth and acid production of mutans streptococci. On the other hand, the cell-surface hydrophobicity of mutans streptococci was significantly reduced by the presence of CM. Furthermore, insoluble glucan synthesis by the glucosyltransferases from either Streptococcus mutans MT8148R or Strep. sobrinus 6715 was inhibited by CM, but not significantly. Hence, the sucrose-dependent cell adherence of mutans streptococci was also depressed by CM. Finally, CM in both a 40% sucrose diet and drinking water resulted in reductions of caries development and plaque accumulation in rats infected with Strep. sobrinus 6715, but not significantly. These results indicate that cacao mass extract possesses some anticariogenic potential, but its anticaries activity is not strong enough to suppress significantly the cariogenic activity of sucrose.

Animals↗
PubMed2002

Proanthocyanidin glycosides and related polyphenols from cacao liquor and their antioxidant effects.

Purification of polar fractions from cacao liquor extracts gave 17 phenolics including four new compounds. The new compounds were characterized as a C-glycosidic flavan, an O-glycoside of a dimeric and two O-glycosides of trimeric A-linked proanthocyanidins, on the basis of spectroscopic data. Isolated polyphenols showed inhibitory effects on nicotinamide adenine dinucleotide phosphate-dependent lipid peroxidation in microsomes and on the autoxidation of linoleic acid. These effects were attributed to the radical-scavenging activity in the peroxidation chain reactions, based on the findings that the cacao polyphenols effectively scavenged the 1,1-diphenyl-2-picrylhydrazyl radical.

Animals↗
PubMed2001

Early detection of cacao swollen shoot virus using the polymerase chain reaction.

A polymerase chain reaction assay was developed which allows early detection of cacao swollen shoot virus (CSSV) in DNA extracts from cacao plantlets agroinoculated with the Togolese isolate Agou 1. The primers used were derived from badnavirus conserved sequences and nucleic acid was extracted with the Plant DNeasy extraction kit (Qiagen). CSSV genome was detectable between 6 and 20 days after inoculation. The first leaf symptoms appeared after 4 weeks and the first shoot swelling symptoms after 8 weeks.

Badnavirus↗
PubMed2005

NEP1 orthologs encoding necrosis and ethylene inducing proteins exist as a multigene family in Phytophthora megakarya, causal agent of black pod disease on cacao.

Phvytophthora megakarya is a devastating oomycete pathogen that causes black pod disease in cacao. Phytophthora species produce a protein that has a similar sequence to the necrosis and ethylene inducing protein (Nep1) of Fusarium oxysporum. Multiple copies of NEP1 orthologs (PmegNEP) have been identified in P. megakarya and four other Phytophthora species (P. citrophthora, P. capsici, P. palmivora, and P. sojae). Genome database searches confirmed the existence of multiple copies of NEP1 orthologs in P. sojae and P. ramorum. In this study, nine different PmegNEP orthologs from P. megakarya strain Mk-1 were identified and analyzed. Of these nine orthologs, six were expressed in mycelium and in P. megakarya zoospore-infected cacao leaf tissue. The remaining two clones are either regulated differently, or are nonfunctional genes. Sequence analysis revealed that six PmegNEP orthologs were organized in two clusters of three orthologs each in the P. megakarya genome. Evidence is presented for the instability in the P. megakarya genome resulting from duplications, inversions, and fused genes resulting in multiple NEP1 orthologs. Traits characteristic of the Phytophthora genome, such as the clustering of NEP1 orthologs, the lack of CATT and TATA boxes, the lack of introns, and the short distance between ORFs were also observed.

Amino Acid Sequence↗
PubMed2001

Developmental variation of sugars, carboxylic acids, purine alkaloids, fatty acids, and endoproteinase activity during maturation of Theobroma cacao L. seeds.

The changes of mono- and oligosaccharides, carboxylic acids, purine alkaloids, and fatty acid composition, and of aspartic endoproteinase activity, were analyzed during seed development in two varieties of cacao (Theobroma cacao). The majority of the components examined either decreased or accumulated steadily in concentration during the second half of bean development. Sucrose is the major sugar in the mature embryo, whereas fructose and glucose are at higher concentrations in the endosperm tissue. Considerable amounts of malate are found in the endosperm, whereas citrate is the dominant carboxylic acid in the embryo. A major change in the fatty acid composition occurs in the young embryo when the proportion of stearic acid increases rapidly at the expense of linoleic acid, which is reduced from about 18 to 3%. Theobromine is the dominant purine alkaloid (ca. 80%), and caffeine appears only toward the end of seed maturity. Aspartic endoproteinase activity increases rapidly during embryo expansion, reaching a maximal activity before final maturity. The results are discussed in conjunction with physiological changes in developing seeds, and the potential contributions of the compounds analyzed for cocoa quality.

Aspartic Acid Endopeptidases↗
PubMed2000

Lipid composition of wild ecuadorian Theobroma subincanum Mart. seeds and comparison with two varieties of Theobroma cacao L.

The present work analyzes the lipid fraction from seeds of wild Ecuadorian Theobroma subincanum and selected commercial varieties of Theobroma cacao from Mexico (var. Criollo) and Ecuador (var. Arriba). The lipid fraction was obtained from the seeds through supercritical fluid extraction and analysis performed by preparatory thin-layer chromatography followed by gas chromatography. The results revealed that in T. subincanum the triglycerides contain fatty acids with longer chains. The melting point and peroxide and saponifiable numbers were determined for each Theobroma sample. The results lead to the conclusion that T. subincanum would produce a poorer quality butter than T. cacao. Nevertheless, the results do point toward a significant commercial use of T. subincanum for low-profile products.

Cacao↗
PubMed2002

Cacao usage by the earliest Maya civilization.

The Maya archaeological site at Colha in northern Belize, Central America, has yielded several spouted ceramic vessels that contain residues from the preparation of food and beverages. Here we analyse dry residue samples by using high-performance liquid chromatography coupled to atmospheric-pressure chemical-ionization mass spectrometry, and show that chocolate (Theobroma cacao) was consumed by the Preclassic Maya as early as 600 bc, pushing back the earliest chemical evidence of cacao use by some 1,000 years. Our application of this new and highly sensitive analytical technique could be extended to the identification of other ancient foods and beverages.

Archaeology↗
PubMed2004

Biosynthesis, accumulation and degradation of theobromine in developing Theobroma cacao fruits.

We have studied the purine alkaloid content and purine metabolism in Theobroma cacao fruits at differing growth stages: Stage A (young small fruit, fresh weight, ca. 2 g); stage B (medium size fruit, fresh weight, ca. 100 g) and stage C (large size, fresh weight, ca. 500 g). The major purine alkaloid in stage A fruits (mainly pericarp) was theobromine (0.7 micromol g(-1) fresh weight), followed by caffeine (0.09 micromol g(-1) fresh weight). The theobromine content of the pericarp decreased sharply with tissue age, and the caffeine content decreased gradually. A large amount of theobromine (22 micromol g(-1) fresh weight) had accumulated in seeds (mainly cotyledons) of stage C fruits. Theobromine was found also in the seed coat and placenta. Tracer experiments with [8-(14)C]adenine show that the major sites of theobromine synthesis are the young pericarp and cotyledons of T. cacao fruits. Limited amounts of purine alkaloids may be transported from the pericarp to seed tissue, but most purine alkaloids that accumulated in seeds appeared to be synthesised in cotyledons. Degradation of [8-(14)C]theobromine and [8-(14)C]caffeine to CO2 via 3-methylxanthine and ureides (allantoin and allantoic acid) was detected only in the pericarp of stage C fruits.

Alkaloids↗