Taira Miyahara

Sequence Analysis of the Genome of Carnation (Dianthus caryophyllus L.)

The whole-genome sequence of carnation (Dianthus caryophyllus L.) cv. ‘Francesco’ was determined using a combination of different new-generation multiplex sequencing platforms. The total length of the non-redundant sequences was 568 887 315 bp, consisting of 45 088 scaffolds, which covered 91% of the 622 Mb carnation genome estimated by k-mer analysis. The N50 values of contigs and scaffolds were 16 644 bp and 60 737 bp, respectively, and the longest scaffold was 1 287 144 bp. The average GC content of the contig sequences was 36%. A total of 1050, 13, 92 and 143 genes for tRNAs, rRNAs, snoRNA and miRNA, respectively, were identified in the assembled genomic sequences. For protein-encoding genes, 43 266 complete and partial gene structures excluding those in transposable elements were deduced. Gene coverage was ∼98%, as deduced from the coverage of the core eukaryotic genes. Intensive characterization of the assigned carnation genes and comparison with those of other plant species revealed characteristic features of the carnation genome. The results of this study will serve as a valuable resource for fundamental and applied research of carnation, especially for breeding new carnation varieties. Further information on the genomic sequences is available at http://carnation.kazusa.or.jp.

The Role of Acyl-Glucose in Anthocyanin Modifications

Higher plants can produce a wide variety of anthocyanin molecules through modification of the six common anthocyanin aglycons that they present. Thus, hydrophilic anthocyanin molecules can be formed and stabilized by glycosylation and acylation. Two types of glycosyltransferase (GT) and acyltransferase (AT) have been identified, namely cytoplasmic GT and AT and vacuolar GT and AT. Cytoplasmic GT and AT utilize UDP-sugar and acyl-CoA as donor molecules, respectively, whereas both vacuolar GT and AT use acyl-glucoses as donor molecules. In carnation plants, vacuolar GT uses aromatic acyl-glucoses as the glucose donor in vivo; independently, vacuolar AT uses malylglucose, an aliphatic acyl-glucose, as the acyl-donor. In delphinium and Arabidopsis, p-hydroxybenzoylglucose and sinapoylglucose are used in vivo as bi-functional donor molecules by vacuolar GT and AT, respectively. The evolution of these enzymes has allowed delphinium and Arabidopsis to utilize unique donor molecules for production of highly modified anthocyanins.

Isolation of an acyl-glucose-dependent anthocyanin 7-O-glucosyltransferase from the monocot Agapanthus africanus.

A cDNA encoding an acyl-glucose-dependent anthocyanin 7-O-glucosyltransferase (AaAA7GT) was isolated from Agapanthus africanus petals; this is the first AAGT identified in a monocot. Peak expression of AaAA7GT in developing A. africanus petals occurred before the flowering stage, and was later than found previously for other anthocyanin biosynthetic genes. Analysis of recombinant proteins showed AaAA7GT had strict substrate preference for anthocyanidin 3-O-glycosides. The AaAA7GT amino acid had high sequence similarity to glycoside hydrolase family 1 (GH1) proteins, which typically act as β-glycosidases. A phylogenetic analysis of amino acid sequences suggested that AAGTs were derived from glycosidase early in the angiosperm lineage.

Repressed expression of a gene for a basic helix-loop-helix protein causes a white flower phenotype in carnation

In a previous study, two genes responsible for white flower phenotypes in carnation were identified. These genes encoded enzymes involved in anthocyanin synthesis, namely, flavanone 3-hydroxylase (F3H) and dihydroflavonol 4-reductase (DFR), and showed reduced expression in the white flower phenotypes. Here, we identify another candidate gene for white phenotype in carnation flowers using an RNA-seq analysis followed by RT-PCR. This candidate gene encodes a transcriptional regulatory factor of the basic helix-loop-helix (bHLH) type. In the cultivar examined here, both F3H and DFR genes produced active enzyme proteins; however, expression of DFR and of genes for enzymes involved in the downstream anthocyanin synthetic pathway from DFR was repressed in the absence of bHLH expression. Occasionally, flowers of the white flowered cultivar used here have red speckles and stripes on the white petals. We found that expression of bHLH occurred in these red petal segments and induced expression of DFR and the following downstream enzymes. Our results indicate that a member of the bHLH superfamily is another gene involved in anthocyanin synthesis in addition to structural genes encoding enzymes.

Carnation I locus contains two chalcone isomerase genes involved in orange flower coloration

Carnations carrying a recessive I gene show accumulation of the yellow pigment chalcononaringenin 2′-glucoside (Ch2′G) in their flowers, whereas those with a dominant I gene do accumulation the red pigment, anthocyanin. Although this metabolic alternative at the I gene could explain yellow and red flower phenotypes, it does not explain the development of orange flower phenotypes which result from the simultaneous accumulation of both Ch2′G and anthocyanin. The carnation whole genome sequencing project recently revealed that two chalcone isomerase genes are present, one that is consistent with the I gene (Dca60979) and another (Dca60978) that had not been characterized. Here, we demonstrate that Dca60979 shows a high level of gene expression and strong enzyme activity in plants with a red flower phenotype; however, functional Dca60979 transcripts are not detected in plants with an orange flower phenotype because of a dTdic1 insertion event. Dca60978 was expressed at a low level and showed a low level of enzyme activity in plants, which could catalyze a part of chalcone to naringenin to advance anthocyanin synthesis but the other part remained to be catalyzed chalcone to Ch2′G by chalcone 2′-glucosyltransferase, resulting in accumulation of anthocyanin and Ch2′G simultaneously to give orange color.

A double knockout mutant of acyl-glucose-dependent anthocyanin glucosyltransferase genes in Delphinium grandiflorum

Blue coloration in delphinium flowers arises from 7-polyacylated anthocyanins which are modified alternately with acyl and glucosyl residues at the 7 position of the aglycone. Previously, we identified two independent genes for acyl-glucose-dependent anthocyanin 7-(6-(p-hydroxybenzoyl)-glucoside) glucosyltransferases (AA7BG-GT); recombinant proteins from the two cDNAs were produced in Escherichia coli and showed AA7BG-GT activity in vitro. Here, a double knockout mutant of both genes was found to lack modification of the second glucosyl residue following further acyl and glucosyl modifications. Both genes in the double mutant had nucleotide sequence changes and deletions that disrupted their transcripts and caused loss of AA7BG-GT activity in sepals. These results provide genetic confirmation that both genes are responsible for AA7BG-GT enzyme activity.