The variability of nuclear DNA content of different Pelargonium species estimated by flow cytometry.

Pelargonium is a versatile genus mainly from the Cape Region, South Africa. The genus is divided into four subgenera and 16 sections characterized by several groups of chromosomes sizes and numbers. The DNA content of species from all subgenera and sections of Pelargonium, except for the sections Subsucculentia and Campylia was estimated using flow cytometry. Nuclei of Pelargonium samples (leaf or petal tissue) and an internal plant standard (leaf tissue) were isolated together and stained with propidium iodide. The DNA content was estimated providing that the 2C peaks of sample and standard be in linearity in the flow cytometer histograms. In total, 96 Pelargonium accessions of 60 species (22 Pelargonium species for the first time) were analyzed. The 2C DNA content ranged from 0.84 pg (P. longifolium, section Hoarea) to 6.69 pg (P. schizopetalum, section Magnistipulacea) and the corresponding 1Cx DNA content from 0.42 pg (P. longifolium) to 1.72 pg (P. transvaalense. This demonstrates the high plasticity within the genus Pelargonium. Some species, such as P. peltatum accessions revealed a pronounced endopolyploidization in leaves but not in petals underlining the importance to choose the right tissue as sample for the flow cytometry analysis. The reported genome sizes are a step forward towards the characterization of the Pelargonium collection within the German Gene Bank for Ornamental Plants and a valuable base for future sequencing programs of the Pelargonium genomes.

Female self-incompatibility type in heterostylous Primula is determined by the brassinosteroid-inactivating cytochrome P450 CYP734A50.

Most flowering plants are hermaphrodites, with flowers having both male and female reproductive organs. One widespread adaptation to limit self-fertilization is self-incompatibility (SI), where self-pollen fails to fertilize ovules.1,2 In homomorphic SI, many morphologically indistinguishable mating types are found, although in heteromorphic SI, the two or three mating types are associated with different floral morphologies.3-6 In heterostylous Primula, a hemizygous supergene determines a short-styled S-morph and a long-styled L-morph, corresponding to two different mating types, and full seed set only results from intermorph crosses.7-9 Style length is controlled by the brassinosteroid (BR)-inactivating cytochrome P450 CYP734A50,10 yet it remains unclear what defines the male and female incompatibility types. Here, we show that CYP734A50 also determines the female incompatibility type. Inactivating CYP734A50 converts short S-morph styles into long styles with the same incompatibility behavior as L-morph styles, and this effect can be mimicked by exogenous BR treatment. In vitro responses of S- and L-morph pollen grains and pollen tubes to increasing BR levels could only partly explain their different in vivo behavior, suggesting both direct and indirect effects of the different BR levels in S- versus L-morph stigmas and styles in controlling pollen performance. This BR-mediated SI provides a novel mechanism for preventing self-fertilization. The joint control of morphology and SI by CYP734A50 has important implications for the evolutionary buildup of the heterostylous syndrome and provides a straightforward explanation for why essentially all of the derived self-compatible homostylous Primula species are long homostyles.11.

Isolation of dihydroflavonol 4-reductase cDNA clones from Angelonia x angustifolia and heterologous expression as GST fusion protein in Escherichia coli.

Blue Angelonia × angustifolia flowers can show spontaneous mutations resulting in white/blue and white flower colourations. In such a white line, a loss of dihydroflavonol 4-reductase (DFR) activity was observed whereas chalcone synthase and flavanone 3-hydroxylase activity remained unchanged. Thus, cloning and characterization of a DFR of Angelonia flowers was carried out for the first time. Two full length DFR cDNA clones, Ang.DFR1 and Ang.DFR2, were obtained from a diploid chimeral white/blue Angelonia × angustifolia which demonstrated a 99% identity in their translated amino acid sequence. In comparison to Ang.DFR2, Ang.DFR1 was shown to contain an extra proline in a proline-rich region at the N-terminus along with two exchanges at the amino acids 12 and 26 in the translated amino acid sequence. The recombinant Ang.DFR2 obtained by heterologous expression in yeast was functionally active catalyzing the NADPH dependent reduction of dihydroquercetin (DHQ) and dihydromyricetin (DHM) to leucocyanidin and leucomyricetin, respectively. Dihydrokaempferol (DHK) in contrast was not accepted as a substrate despite the presence of asparagine in a position assumed to determine DHK acceptance. We show that substrate acceptance testing of DFRs provides biased results for DHM conversion if products are extracted with ethyl acetate. Recombinant Ang.DFR1 was inactive and functional activity could only be restored via exchanges of the amino acids in position 12 and 26 as well as the deletion of the extra proline. E. coli transformation of the pGEX-6P-1 vector harbouring the Ang.DFR2 and heterologous expression in E. coli resulted in functionally active enzymes before and after GST tag removal. Both the GST fusion protein and purified DFR minus the GST tag could be stored at -80°C for several months without loss of enzyme activity and demonstrated identical substrate specificity as the recombinant enzyme obtained from heterologous expression in yeast.