The Light Response Of COP1 And SPA And Their Roles In Fragrance Biosynthesis Of Lilium 'Siberia'

Lilium ‘Siberia' is one of the most common cultural Lilium Oriental hybrids, as a perennial herb, Lilium ‘Siberia' is a popular ornamental plant for its flowers possessing intense fragrance,wonderful shape and longevity,that's why it widely used as the world's most famous cut flowers. The unique combination of floral scent molecules emitted by flowers is determined by combination of low molecular weight compounds, which mainly constituted by phenylpropanes and terpenoids incluing ethyl benzoate, linalool, ocimene etc.In this research, we uses the Lilium ‘Siberia' cut flowers as material under different light treatments and studied by the methods of RT-PCR, subcellular localization,yeast two-hybrid,Bifc, transient overexpression and surppression,q RT-PCR and GC-MS. Light is an important environmental signal that governs plant growth, development and metabolism,One key light-signaling component involved in plant light responses is constitutively photomorphogenic 1(COP1).Lo COP1 protein is not only a Photomorphogenesis suppressor, but also a molecular switch for light control in plant development. We explore light response of Lo COP1 and its interacting factors Lo SPA and Lo MYBs in the regulation of rhythmical fragrance emission of Lilium ‘Siberia'. These results will shed some light on the theoretical basis for molecular mechanisms on the formation and rhythmical fragrance emission in Lilium. The main results were as follows:1. Determination of light and dark treatment effects on the fragrance and its contents were measured by GC-MS method. The results showed that under the dark and red light treatments can significantly reduce the release of four main chemical components(the ethyl benzoate, linalool, ocimene and alloocimene) in ‘Siberia'. In addition, the emission of terpenoid aromatic components(linalool, ocimene and alloocimene) of ‘Siberia'- were rhythmical no effect in the light or dark condition, but under continuous dark condition, this rhythm of Phenylaprapanoid ethylbenzoate collapsed, and its content decreased significantly. Moreover, the fragrance emission obviously relys on the light, Within four hours, the plant can respond quickly to the changes of light.2. According to transcriptome data, we cloned the cDNA sequence of Lo COP1 and Lo SPA. The cDNA length of COP1 was 2067 bp and coded 689 amino acids protein, whichconsisted of three special structural domains: an annular zinc finger domain, coiled-coil region and WD-40 repeat sequences, the cDNA length of SPA is 2676 bp and they code 892 amino acids protein. RT-q PCR analysis showed that Lo COP1 and Lo SPA both were expressed at highest level in sepal, followed by the petal, and lowest level in the roots.With the opening of flowers, the Lo COP1 expression in sepal gradually increased then decreased slightly, but in petal, Lo COP1 expressed in every period have no significant change. Lo SPA in the petals and sepals of the half-open in the early opening of the expression is slightly higher.3. Lo COP1-GFP and Lo SPA-GFP in onion epidermal cells formed green speckles in nuclear and under DAPI light showed blue speckles, indicating that Lo COP1 and Lo SPA located in nucleus.under 48 hours continue light or dark, Green fluorescence of Lo COP1 was clearly detectable with combinations of proteins no matter in dark or light condition,while more green speckles are clearly observed in the continue dark than light condition,meanwhile the number of fluorescent nuclei under different conditions have tremendous differences, suggesting that Lo COP1 is able to translocate in a light-dependent manner.4. The yeast two hybrid assaies were carried out to demonstrate that Lo COP1 can interact with Lo SPA, Lo MYB1, Lo MYB3 respectively, Lo SPA can interact with Lo MYB1,Lo MYB2, Lo MYB3. The results from ?-galactosidase assays further confirmed those results. Additionly, Bi FC results suggesting that the R2R3-MYB transcription factors Lo MYB1 and Lo MYB3 were found as interactors of Lo COP1 protein, Lo MYB1, Lo MYB2 and Lo MYB3 were as interactors of Lo SPA protein and Lo COP1 and Lo SPA can form a complex in vivo in plant cells. Moreover Lo COP1 and Lo SPA interacts with Lo MYB1 and Lo MYB3 in onion epidermal cells controlled by light.5. To verify the function of Lo COP1 in lily fragrance emission, The BSMV-VIGS vectors of Lo COP1 and Lo SPA were constructed. There were used to transformed into Lilium petals by Agrobacterium. The results reveal that Lilium transfected with BSMV-Lo COP1, the Lo COP1 gene expression fell down to 42.2 %, indicating the BSMV was effective in the Lilium. Ocimene, linalool, ethyl benzoate, methyl benzoate were increased of 2.9 fold, 0.9 fold, 0.4 fold, 0.2 fold respectively. Lo SPA gene expression fell down to 43.9 %, ocimene, linalool, ethyl benzoate, methyl benzoate were increased of 5.9fold, 3.5 fold, 4.0 fold, 2.0 fold respectively.6. The p OX vector of Lo COP1 was constructed. It was transformed into Lilium petals by Agrobacterium.The result showed that after instantaneous overexpression of Lo COP1,the expression of Lo COP1 rose about 37.6 %,coupling with MYB1,MYB2,MYB3,AAT and TPS gene expression descreased about 88.5 %, 94.6 % and 41.4 %,99.4 % and 93.2 %respectively. ocimene, linalool, ethyl benzoate, methyl benzoate were declined of 60.4 %,58.8 %, 76.4 %, 89.3 %.The overexpression of Lo COP1 inhibited the emission of fragrance and suppression of Lo COP1 promoted the emission of fragrance, indicating that Lo COP1 played a negative regulatory role in the synthesis of fragrance emission.