Study On The Effect And Mechanism Of ALA-regulated Anthocyanin And Flavonol Accumulation In Apple Callus

Anthocyanins,which play an important role in fruit apperaence quality,influence the consumers if buy the fruit or not,and also affect the economic value of fruits.Flavonols,as another important kind of flavonoids,are reported to do a great job in plant stress resistance,colour formation and regulation of plant growth and development.5-Aminolevulinic acid(ALA),is a new plant growth regulator.It has been reported that ALA can promote the accumulation of anthocyanins in apple peels and affect the fruit appearance quality.At the same time,it also promotes the accumulation of flavonols,facilitate the removal of reactive oxygen species,regulate stomatal movement in leaves,and improve plant resistance to abiotic stresses.However,how ALA regulates the biosynthesis of these two kinds of metabolites needs further study.Therefore,using ’Fuji’ apple callus(Malus × domenstica Borkh.cv.Fuji)are as the plant materials in this experiment.We studied the interaction between the apple transcription factor MdMADSl and the promoter of the key biosynthetic genes of anthocyanin.On the other hand,the positive effect on apple flavonol synthesis induced by ALA was determined.And a new mechanism for the regulation of apple flavonol biosynthesis was also revealed.The main results are as follows.1.Based on the previous research in our lab,the transcription factor MdMADS1 plays an important role in the accumulation of anthocyanin induced by ALA in apple.To reveal the function mechanism of MdMADSl,here,yeast one-hybrid technology was used to study the combination of MdMADS1 with promoters of MdCHS,MdDFR,MdLDOX,MdUFGT,four key biosynthetic genes of anthocyanin.The bait sequence consists of CArG sequence 5’-CC(A/T)6GG-3’,N10-like sequence 5’-CTA(A/T)4TAG-3’ or CARGCW8GAT sequences 5’-C(A/T)8G-3’ in the promoter region of these genes.The results of yeast one-hybrid experiments revealed that the transcription factor MdMADS1 could bind to the N10-like sequence of the MdCHS and MdUFGT promoters.No clear interaction with CArG sequence.Subsequently,three mutant sequences were designed based on the predicted N10-like sequence 5’-CTATTTATAG-3’ in the MdUFGT promoter.The yeast one-hybrid result of these mutant baits indicate that the core DNA binding site of MdMADS1 is 5’-CTATAG-3’.In addition,we verified that MdMADS1 can up-regulate the expression of MdCHS and MdUFGT genes by using dual luciferase reporter gene assays.2.To understand the regulatory mechanism behind ALA-promoted flavonol accumulation,we first analyzed the transcriptome data of previous study in our lab and found that five MdFLS genes were significantly induced by ALA,and then one of these five MdFLS genes was chosen and obtained the transgenic apple callus of pMdFLS1::GUS.By treated transgenic callus with ALA,we observed the promotive effect of ALA on MdFLS1 promoter activity.This indicates that ALA can promote the activity of MdFLS1 promoter,a key gene of apple flavonol synthesis,promote its gene expression and induce flavonol accumulation.3.To further understand the regulatory mechanism of apple flavonol biosynthesis.In this section,the apple callus cDNA library is first established,and then using the MdFLS1 promoter sequence as the bait sequence,we screened apple callus cDNA library.Results showed that a total of 135 positive clones were screened.After the yeast colony PCR and sequencing analysis,some false positive and housekeeping genes were removed,and 23 proteins that could interact with the MdFLS1 promoter were identified.Among them,the transcription factor MdSCL8 was chosen,and further bioinformatic analysis and function prediction were performed.These above findings in our study open up new horizons for exploring the molecular mechanism of ALA regulating the synthesis of anthocyanins and flavonols,and also provide a theoretical basis for ALA to improve apple fruit coloring and enhance plant stress resistance.