The Role Of Hexose And Hexokinase In Fragrance Biosynthesis Of Lilium 'siberia'

Floral scent is a very valuable ornamental trait,which is very essential for the study of the synthesis mechanism for improving the floral traits of plants.In plants,sugar is an important energy source for plant metabolism and its content can significantly regulate plant growth and development.Phosphorylation of hexoses is an important step in sugar metabolism in plants.Hexokinase phosphorylation by hexoses has the dual function of regulating metabolic flux and serving as a sugar signal,but there are few studies on the role of hexose and hexokinase genes in the metabolic regulation of floral fragrance biosynthesis.It is of great scientific significance to explore whether it modulates floral floral fragrance biosynthesis by catalyzing hexose and signaling as a sugar sensor protein.In this study,Lilium‘Siberia'cut flowers were used as research material treated by exogenous fructose and glucose respectively,and combination with the HXK inhibitor N-acetyl-glucosamine?NAG?in full-dark condition.By GC-MS technique,subcellular localization,yeast two-hybrid,bimolecular fluorescence complementation,transient overexpression and silencing and RT-qPCR the roles of hexose and hexokinase in fragrance biosynthesis and release in lily flowers were investigated.The main results were as follows:1.GC-MS technique was used to determine the endogenous soluble sugar and circadian rhythm of the petals of Lilium‘Siberia'in natural light.The results showed that the petals of'Siberia'mainly had three kinds of soluble sugars including fructose,sucrose and glucose,the contents of fructose and glucose were high and showed circadian rhythm,but sucrose content was low and showed no obvious circadian rhythm.2.The effects of fructose,glucose,HXK inhibitors NAG,NAG and glucose cocktails on biosynthesis and release of fragrance in‘Siberia'were analysed by GC-MS in full-dark condition.The results showed that both fructose and glucose can significantly increase the content of terpenes including ocimene and linalool.After treatment with NAG,the promotion effect of glucose?ocimene and linalool?was obviously inhibited.But neither of them affected the synthesis of ethyl benzoate.At the same time,the release of the terpenes still had the same apparent circadian rhythm as in natural light,but the release rhythm of ethyl benzoate was disrupted.3.According to the transcriptome data of Lilium‘Siberia',the full-length cDNA sequences of the two hexokinases were cloned by RT-PCR and named as LoFRK and LoHXK according to their substrate affinities.The cDNA length of LoFRK is 996bp encoding a protein of 332 amino acids and the cDNA length of LoHXK is 1500bp encoding a protein of 500 amino acids.After treatment with fructose and glucose,the expression levels of LoFRK,LoHXK and terpene synthetases LoTPS1 and LoTPS3 were significantly increased,and the expression trend was consistent with the emission of floral fragrance.RT-qPCR analysis showed that both LoFRK and LoHXK genes were expressed in eight different tissues and organs,including anthers,pistils,petals,leaves and stems,and moreover they were all expressed in anthers.In the meantime with the opening of flowers,the expression level of LoFRK was increased and showed highest in the decline period.The expression level of LoHXK was no significant difference among different flower development stage.4.A full-length GFP fusion expression vectors of LoFRK and LoHXK were constructed and used nuclear marker and mitochondrial marker to expression in Arabidopsis protoplasts in the same time.The results indicated that LoFRK located in cytoplasm and nucleus,while LoHXK located in mitochondria.5.Yeast double-hybrid experiments were conducted by constructing LoFRK and LoHXK yeast two-hybrid bait vectors and using the LoMYBs and LoMYC capture vectors that have been constructed in the laboratory.The experiment showed that LoFRK can interact with LoMYB1 and LoMYB2 respectively.And meanwhile in order to eliminate the false positive of yeast two-hybrid,the LoFRK bimolecular fluorescent complementary C-terminal vector?cYFP?and LoMYB1,LoMYB2 N-terminal vector?nYFP?were constructed and both fusion proteins were transformed into onion epidermal cells.After two days,green fluorescence was also observed and blue fluorescence was observed in the cell nucleus after DAPI staining,which showed that LoFRK can interact with LoMYB1and LoMYB2 in the nucleus.6.To analyze the function of hexokinases in lily fragrance emission,The BSMV-VIGS vectors of LoFRK and LoHXK were constructed.It was used to transformed into Lilium petals by Agrobacterium for transient suppression.And the data stated that after the expression level of LoFRK and LoHXK were suppressed,ocimene decreased by 60%and 43%respectively,linalol decreased by 52%and 33%respectively,while ethyl benzoate content did not change significantly.And meanwhile RT-PCR analysis revealed that the expression levels of LoFRK and LoHXK decreased by 61%and 58%,LoTPS1 expression decreased by 70%and 80%,LoTPS3 expression decreased by 79%and 87%,LoMYB1expression decreased by 35%and 75%,LoMYB2 expression decreased by 78%and 39%,respectively.7.The major floral components in mass spectrogram after GC-MS determination of Lilium in ¹³C-labeled fructose and glucose were analyzed under full darkness.The results indicated that the mass spectrogram of the linalool and ocimene had changed significantly after ¹³C-labeled fructose and glucose and the m/z was increased but ethyl benzoate did not change visibly.The results indicated that exogenous fructose and glucose as substrates only catalyzed the synthesis of linalool and ocimene and did not participate in the synthesis of ethyl benzoate,a phenylpropanoid volatile substance.8.The LoFRK and LoHXK catalytic sites mutation and unmutated overexpression vectors were constructed.The LoFRK and LoHXK recombinant vectors were infected by Agrobacterium for transient overexpression of the‘Siberian‘petals.the experiment demonstrated that compared with no-load,the mutant catalytic sites and LoFRK overexpression,the increase in the ocimene and linalool were 65%and 79%.However,compared to the unmutated enzyme and overexpression LoFRK,ocimene and linalool were respectively lower 77%and 64%after the catalytic enzyme sites.And meanwhile,the experiment demonstrated that compared with no-load,the mutant catalytic sites and LoHXK overexpression,the increase in the ocimene and linalool were 30%and 74%.However,compared to the unmutated enzyme and overexpression LoFRK,ocimene and linalool were respectively lower 91%and 50%after the catalytic enzyme sites.And RT-PCR analysis revealed that after mutation of the catalytic site and unmutated overexpression of LoFRK and LoHXK,LoFRK expression was increased by 75%and 98%,LoHXK expression was increased by 67%and 112%,LoTPS1 was increased by 43%,99%,24%and 56%,LoTPS3was upregulated by 25%,86%,21%and 66%,LoMYB1 was increased by 65%,103%,23%and 30%,LoMYB2 expression was increased by 99%,12%,30%and 83%,respectively.