Fast determination of N-phenylpropenoyl-l-amino acids (NPA) in cocoa samples from different origins by ultra-performance liquid chromatography and capillary electrophoresis.

N-Phenylpropenoyl-l-amino acids (NPA) are among the key contributors to the astringent taste of cocoa. Two fast and easy to use methods (CE and UPLC®, both with PDA detection) for routine determination of the main NPA were developed. Crude extracts of defatted seeds were analysed by means of capillary electrophoresis leading to separation in less than 30min. Separation by means of UPLC® was much faster (<4min), however, a preceding SPE clean-up abolishes this benefit in time saving. Thus, the CE- and UPLC®-methods are comparable concerning time consumption and provide similar results. Analysis of 18 samples of raw and roasted beans from the global cocoa market originated from 12 countries and 4 continents showed a great variability of NPA content (0.7-3.6mg/g) and qualitative composition of different NPA. Anyway, all samples from cocoa beans showed a comparable NPA pattern. N-[3',4'-dihydroxy-(E)-cinnamoyl]-l-aspartic acid was the most abundant metabolite, followed by N-[4'-hydroxy-(E)-cinnamoyl]-l-aspartic acid and N-[3',4'-dihydroxy-(E)-cinnamoyl]-3-hydroxy-l-tyrosine (clovamide). The analysis of other plant organs (flowers, leaves, fruits) revealed an entirely different situation. NPA were detected in all parts of the fruit, with husk and pulp being clearly dominated by clovamide. In flowers and leaves no NPA were detected; 2-O-caffeoyltartaric acid was shown to be the major caffeic acid metabolite in leaves.

Characterization of MADS-box genes in charophycean green algae and its implication for the evolution of MADS-box genes.

The MADS-box genes of land plants are extensively diverged to form a superfamily and are important in various aspects of development including the specification of floral organs as homeotic selector genes. The closest relatives of land plants are the freshwater green algae charophyceans. To study the origin and evolution of land plant MADS-box genes, we characterized these genes in three charophycean green algae: the stonewort Chara globularis, the coleochaete Coleochaete scutata, and the desmid Closterium peracerosum-strigosum-littorale complex. Phylogenetic analyses suggested that MADS-box genes diverged extensively in the land plant lineage after the separation of charophyceans from land plants. The stonewort C. globularis mRNA was specifically detected in the oogonium and antheridium together with the egg and spermatozoid during their differentiation. The expression of the C. peracerosum-strigosum-littorale-complex gene increased when vegetative cells began to differentiate into gametangial cells and decreased after fertilization. These expression patterns suggest that the precursors of land plant MADS-box genes originally functioned in haploid reproductive cell differentiation and that the haploid MADS-box genes were recruited into a diploid generation during the evolution of land plants.

Two ancient classes of MIKC-type MADS-box genes are present in the moss Physcomitrella patens.

Characterization of seven MADS-box genes, termed PPM1-PPM4 and PpMADS1-PpMADS3, from the moss model species Physcomitrella patens is reported. Phylogeny reconstructions and comparison of exon-intron structures revealed that the genes described here represent two different classes of homologous, yet distinct, MIKC-type MADS-box genes, termed MIKC(c)-type genes-"(c)" stands for "classic"-(PPM1, PPM2, PpMADS1) and MIKC(*)-type genes (PPM3, PPM4, PpMADS2, PpMADS3). The two gene classes deviate from each other in a characteristic way, especially in a sequence stretch termed intervening region. MIKC(c)-type genes are abundantly present in all land plants which have been investigated in this respect, and give rise to well-known gene types such as floral meristem and organ identity genes. In contrast, LAMB1 from the clubmoss Lycopodium annotinum was identified as the only other MIKC(*)-type gene published so far. Our findings strongly suggest that the most recent common ancestor of mosses and vascular plants contained at least one MIKC(c)-type and one MIKC(*)-type gene. Our studies thus reveal an ancient duplication of an MIKC-type gene that occurred before the separation of the lineages that led to extant mosses and vascular plants more than about 450 MYA. The identification of bona fide K-domains in both MIKC(*)-type and MIKC(c)-type proteins suggests that the K-domain is more ancient than is suggested by a recent alternative hypothesis. MIKC(*)-type genes may have escaped identification in ferns and seed plants so far. It seems more likely, however, that they represent a class of genes which has been lost in the lineage which led to extant ferns and seed plants. The high number of P. patens MADS-box genes and the presence of a K-box in the coding region and of some potential binding sites for MADS-domain proteins and other transcription factors in the putative promoter regions of these genes suggest that MADS-box genes in mosses are involved in complex gene regulatory networks similar to those in flowering plants.