Effects Of Vernalization On DNA Methylation And Protein Expression In Sugarbeet Sterile Line And Maintainer Line

Sugar Beet(Beta vulgaris ssp.vulgaris)is a biennial,sucrose-storing plant.To induce bolting(i.e.rapid stem elongation followed by flowering),sugar beet requires a prolonged exposure to low temperatures between 2 °C and 10 °C.On the one hand,vernalization can accelerate the breeding process of sugar beet.On the other hand,early bolting drastically reduces the beet and sugar yield.At present,the flowering mechanism of sugar beet hasn't been clarified yet.Cytosine methylation is a well recognized epigenetic mark,while its effects on the regulation of beet vernalization is still under study.Vernalization is a long-term process.Many studies have shown that the responses to low temperatures leading to cold acclimation and vernalization are controlled by distinct signalling pathways,but their differences at protein level were still poorly studied.In this paper,the methylation status and transcript abundance of 4 genes(BvFT1,BvFT2,BvCPSF73-Ia,and BvCPSF73-Ib)that response to vernalization and protein expression in the process of low temperature treatment were explored in different fertility sugar beet cultivars.The main contents and results are as follows:1.Sugar beet seedlings were treated at 8 ? for vernalization and 25 ? for control,respectively.Vernalized beet roots were treated at 8 ? for vernalization and 25 ? for devernalization,respectively.Mass Array flight mass spectrometry analysis system was used to detect the cytosine methylation of vernalization-related genes.The results showed that the methylation status of DNA changed dynamically with the growth of plant.The methylation mode of DNA was different under different temperature conditions,and the methylation status in the plant could be remodeled by vernalization at low temperature and devernalization at high temperature.There were differences in DNA methylation between male sterile line and maintainer line.The methylation level of multiple loci was polymorphic in four vernalization related genes.2.Using the same material as methylation detection,we used fluorescence quantitative PCR to detect gene expression.In Seedlings,BvFT1 and BvFT2 had the opposite trend of expression,which confirms the inhibition of BvFT1 on BvFT2,but they didn't follow this trend completely in roots,indicating that there may be other factors to regulate the expression of BvFT2 gene except BvFT1.The expression of BvCPSF73-Ia and BvCPSF73-Ib was increased at low temperature in seedlings.In roots,the expression of BvCPSF73-1a gene in the vernalization group at the end of treatment was higher than that in the devernalization group.The BvCPSF73-1b gene was the opposite,probably because the CPSF73 protein was necessary for the plant development.3.In order to further clarify the effect of DNA methylation on gene expression,the correlation analysis of the methylation rate at each CG site and relative expression of genes was analyzed.In seedlings,there was significant positive correlations between the DNA methylation rate at the-718 site of the BvFT2 and the relative expression amounts.In roots,there was significant positive correlations between the DNA methylation rate at the-1637 site of the BvFT1 and the relative expression amounts.4.The beet seedlings in the same growing state were treated with cold acclimation and long-term vernalization at 8 ?.Proteomic analysis was carried out by iTRAQ technology.The difference in expression of protein increased gradually with the prolongation of cold treatment,and the number of differential proteins between the male sterile lines and the maintainers increased gradually.Under cold acclimation,the differentially expressed proteins in male sterile line and maintainer line were mainly related to photosynthesis.Under vernalization conditions,the differentially expressed proteins in sterile lines and maintainers were mainly derived from ribosomes and mitochondria.Compared proteins under vernalization and cold acclimation conditions,these differentially expressed proteins mainly related to photosynthesis,respiration,reproductive growth and stress response.There were significant differences in the expression of methylase between sterile line and maintainer line under low temperature stress.