Download 題目: Regulatory mechanism of floral coloration in Oncidium cultivars

題目: Regulatory mechanism of floral coloration in Oncidium cultivars
作者：邱崇益 Chung-Yi Chiou
指導老師：葉開溫
D03
Abstract
Oncidium are among the most important plants in the flower market in Taiwan,
however little research on pigmentation biosynthetic genes has been reported.
Therefore, the isolation of pigmentation biosynthesis related genes and further study
color patterning in floral tissue of Oncidium Gower Ramsey and carotenoid-related
genes determines diversified carotenoid coloration in Oncidium Gower Ramsey
“Sunkist” and Oncidium Gower Ramsey “White Jade”
In this study, chromatography analysis revealed that the red coloration in floral
tissues was composed of malvidin-3-O-galactoside, peonidin-3-O-glucoside,
delphinidin-3-O-glucoside and cyanidin-3-O-glucoside compounds. By contrary,
these pigments were not detected in yellow lip tissue. Four key genes involved in
anthocyanin biosynthetic pathway, i.e. chalcone synthase (OgCHS), chalcone
isomerase (OgCHI), dihydroflavonol 4-reductase (OgDFR) and anthocyanidin
synthase (OgANS) were isolated and their expression patterns were characterized.
Northern blot analysis confirmed that although they are active during floral
development, OgCHI and OgDFR genes are specifically down-regulated in yellow
lip tissue. Bombardment with OgCHI and OgDFR genes into lip tissue driven by a
flower-specific promoter, Pchrc (chromoplast-specific carotenoid-associated gene),
demonstrated that transient expression of these two genes resulted in anthocyanin
production in yellow lip. Further analysis of a R2R3 MYB transcription factor,
OgMYB1, revealed that although it is actively expressed during floral development, it
is not expressed in yellow lip tissue. Transient expression of OgMYB1 in lip tissues by
bombardment can also induce formation of red pigments through the activation of
OgCHI and OgDFR transcription. These results demonstrate that differential
expression of OgMYB1 is critical to determine the color pattern of floral organ in
Oncidium Gower Ramsey. The differential expression of OgMYB1 is caused by DNA
methylation.
Three cultivars of Oncidium orchid with varied coloration, such as Oncidium
Gower Ramsey (yellow), Sunkist (orange) and White Jade (white), were analyzed for
carotenoid metabolites and gene expression of carotenoid-biosynthetic genes. The
HPLC analysis revealed that yellow Gower Ramsey accumulates violaxathin, 9-cisviolaxathin and neoxanthin, orange Sunkist accumulates an additional β-carotene, and
White Jade is devoid of carotenoid compounds. Molecular characterization indicated
that the three Oncidium cultivars exhibited varied expression pattern and level in
carotenoid-biosynthetic pathway. Among them, high expression level of
β-hydroxylase (OgHyb) and zeaxathin epoxidase (OgZep) was displayed in yellow
Gower Ramsey, relative to the down-regulation of OgHyb and OgZep exhibited in
orange Sunkist, which results in the accumulation of β-carotene and orange coloration
in floral tissues. However, White Jade is caused by the up-regulation of CCD1
(Carotenoid Cleavage Dioxygenase 1), which catabolizes carotenoid metabolites.
Methylation assay of CCD1 promoter in White Jade and Gower Ramsey revealed that
a high level of DNA methylation was present in CCD1 promoter region of Gower
Ramsey. Transient expression of OgCCD1 in yellow lip tissues of Gower Ramsey by
bombardment confirmed its function of disintegrating carotenoid compounds. Our
results suggest an evolutionary significance that genetic variation of
carotenoid-related genes in Oncidium generates the complexity of floral pigmentation,
and consequently provides the profound varieties in Oncidium population.
Tissue-specific promoters are required for plant molecular breeding to drive a
target gene in the appropriate location in plants. In this study, a chromoplast specific
carotenoid-associated gene (OgCHRC) and its promoter (Pchrc) were isolated from
Oncidium orchid and characterized. Northern blot analysis revealed that OgCHRC is
specifically expressed in flowers, not in roots and leaves. Transient expression assay
of Pchrc by bombardment transformation confirmed its differential expression pattern
in floral tissues of different horticulture plants and cell-type location in conical
papillate cells of adaxial epidermis of flower. These results suggest that Pchrc could
serve as a useful tool in ornamental plant biotechnology to modify flower color.