Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Genetic engineering of flower color provides biotechnological products such as blue carnations or roses by accumulating delphinidin-based anthocyanins not naturally existing in these plant species. Betalains are another class of pigments that in plants are only synthesized in the order Caryophyllales. Although they have been engineered in several plant species, especially red-violet betacyanins, the yellow betaxanthins have yet to be engineered in ornamental plants. We attempted to produce yellow-flowered gentians by genetic engineering of betaxanthin pigments. First, white-flowered gentian lines were produced by knocking out the dihydroflavonol 4-reductase (DFR) gene using CRISPR/Cas9-mediated genome editing. Beta vulgaris BvCYP76AD6 and Mirabilis jalapa MjDOD, driven by gentian petal-specific promoters, flavonoid 3',5'-hydroxylase (F3'5'H) and anthocyanin 5,3'-aromatic acyltransferase (AT), respectively, were transformed into the above DFR-knockout white-flowered line; the resultant gentian plants had vivid yellow flowers. Expression analysis and pigment analysis revealed petal-specific expression and accumulation of seven known betaxanthins in their petals to c. 0.06-0.08 μmol g FW-1 . Genetic engineering of vivid yellow-flowered plants can be achieved by combining genome editing and a suitable expression of betaxanthin-biosynthetic genes in ornamental plants.

DOI： 10.1111/nph.19218

PubMed

Publishing type：Research paper (scientific journal)  

Gentians cultivated in Japan (Gentiana triflora and Gentiana scabra and hybrids) have blue flowers, but flower colour intensity differs among cultivars. The molecular mechanism underlying the variation in flower colour intensity is unclear. Here, we produced F₂ progeny derived from an F₁ cross of intense- and faint-blue lines and attempted to identify the genes responsible for flower colour intensity using RNA-sequencing analyses. Comparative analysis of flower colour intensity and transcriptome data revealed differentially expressed genes (DEGs), although known flavonoid biosynthesis-related genes showed similar expression patterns. From quantitative RT-PCR (qRT-PCR) analysis, we identified two and four genes with significantly different expression levels in the intense- and faint-blue flower lines, respectively. We conducted further analyses on one of the DEGs, termed GtMIF1, which encodes a putative mini zinc-finger protein homolog, which was most differently expressed in faint-blue individuals. Functional analysis of GtMIF1 was performed by producing stable tobacco transformants. GtMIF1-overexpressing tobacco plants showed reduced flower colour intensity compared with untransformed control plants. DNA-marker analysis also confirmed that the GtMIF1 allele of the faint-blue flower line correlated well with faint flower colour in F₂ progeny. These results suggest that GtMIF1 is one of the key genes involved in determining the flower colour intensity of gentian.

DOI： 10.3389/fpls.2022.906879

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Cultivated Japanese gentians traditionally produce vivid blue flowers because of the accumulation of delphinidin-based polyacylated anthocyanins. However, recent breeding programs developed several red-flowered cultivars, but the underlying mechanism for this red coloration was unknown. Thus, we characterized the pigments responsible for the red coloration in these cultivars. A high-performance liquid chromatography with photodiode array analysis revealed the presence of phenolic compounds, including flavones and xanthones, as well as the accumulation of colored cyanidin-based anthocyanins. The chemical structures of two xanthone compounds contributing to the coloration of red-flowered gentian petals were determined by mass spectrometry and nuclear magnetic resonance spectroscopy. The compounds were identified as norathyriol 6-O-glucoside (i.e., tripteroside designated as Xt1) and a previously unreported norathyriol-6-O-(6'-O-malonyl)-glucoside (designated Xt2). The copigmentation effects of these compounds on cyanidin 3-O-glucoside were detected in vitro. Additionally, an RNA sequencing analysis was performed to identify the cDNAs encoding the enzymes involved in the biosynthesis of these xanthones. Recombinant proteins encoded by the candidate genes were produced in a wheat germ cell-free protein expression system and assayed. We determined that a UDP-glucose-dependent glucosyltransferase (StrGT9) catalyzes the transfer of a glucose moiety to norathyriol, a xanthone aglycone, to produce Xt1, which is converted to Xt2 by a malonyltransferase (StrAT2). An analysis of the progeny lines suggested that the accumulation of Xt2 contributes to the vivid red coloration of gentian flowers. Our data indicate that StrGT9 and StrAT2 help mediate xanthone biosynthesis and contribute to the coloration of red-flowered gentians via copigmentation effects.

DOI： 10.1111/tpj.15412

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Red cabbage anthocyanin is utilized as a natural food colorant because of its stable and brilliant coloration. The major anthocyanin of red cabbage is cyanidin (Cy) mono- and di-acyltriglucoside; however, the biosynthetic pathway to generate this anthocyanin remains unclear. We isolated and identified four uridine diphosphate-glucose-dependent glucosyltransferase (UGT) cDNAs from red cabbage using RNA-seq. UGTs are involved in Cy triglucoside (CytriG) synthesis, the precursor of Cy acyltriglucoside. Enzymatic assays using recombinant proteins suggested that UGT78D5 encodes Cy 3GT, UGT79B45 encodes Cy 3-glucoside GT, UGT75C2 encodes Cy 3-sophoroside (Cy3Sp) 5GT, and UGT79B44 encodes flavonol 3-glucoside GT. Anthocyanin GT assays using crude proteins prepared from red cabbage suggested that CytriG is produced from intermediate products in the following order: Cy, Cy3G, Cy3Sp, and CytriG.

DOI： 10.1021/acs.jafc.0c03480

PubMed

International / domestic magazine：International journal  

Genome editing by the CRISPR/Cas9 system has recently been used to produce gene knockout lines in many plant species. We applied this system to analyze Japanese gentian plants that produce blue flowers because of the accumulation of a polyacylated anthocyanin, gentiodelphin. Mutant lines in which anthocyanin modification genes were knocked out were examined to assess the contribution of each gene to the blue pigmentation of flowers. The targeted genes encoded anthocyanin 5-O-glycosyltransferase (Gt5GT), anthocyanin 3'-O-glycosyltransferase (Gt3'GT), and anthocyanin 5/3'-aromatic acyltransferase (Gt5/3'AT). The Gt5GT knockout lines accumulated delphinidin 3G, whereas the Gt3'GT knockout lines accumulated delphinidin 3G-5CafG as the major flower pigment. Knocking out Gt5/3'AT resulted in the accumulation of delphinidin 3G-5G-3'G and delphinidin 3G-5G as the primary and secondary pigments, respectively. These results indicated the existence of two pathways mediating the modification of delphinidin 3G-5G in flowers, with one involving a glycosylation by 3'GT and the other involving an acylation by 5/3'AT. The Gt5GT, Gt3'GT, and Gt5/3'AT transformants produced pale red violet, dull pink, and pale mauve flowers, respectively, unlike the vivid blue flowers of wild-type plants. Thus, the glycosylation and subsequent acylation of the 3'-hydroxy group of the B-ring in delphinidin aglycone is essential for the development of blue gentian flowers.

DOI： 10.1038/s41598-019-51808-3

PubMed

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Venue：東京