| Hyperhydricity(HH)is a physiological disorder and morphological malformation owing to stresses related to in vitro culture conditions,which often occurs in plant tissue culture.Hyperhydric plantlets show a typical 'glassy' or 'vitrescent' appearance,which is characterized by thick,translucent,curled,and brittle leaves.These plantlets survive very poorly when they are subcultured in fresh medium or transferred to an ex vitro environment.It can cause considerable losses in commercial micropropagation industry by reducing the quality and multiplication rate of the cultured plants.It also limits the application of tissue culture methods to the conservation of plant resources and genetic transformation of plants.Up to now,the molecular mechanism of HH is not yet clear.Therefore,it has important theoretical and practical significance for studying the mechanism of hyperhydricity and screening effective control measures.which paves the way for the sound development of tissue culture technology.In this study,the morphological structure,physiological and biochemical were investigated in D.chinensis L.and Arabidopsis thaliana.In addition,DNA methylation and transcriptome database were established through High-throughput mRNA sequencing(RNA-Seq)and Whole genome bisulfite sequencing(WGBS)to identify differentially expressed and methylated genes between normal and hyperhydric Arabidopsis seedlings.On this basis,the mechanism of ethylene on the occurrence of hyperhyrdricity and the effect of AgNO3 on the control of hyperhyrdricity were studied systematically.The main results are as follows:(1)Scanning electron microscopy(SEM)and transmission electron microscopy(TEM)were used for morphological structure observation of normal and hyperhydric plantlets.Compared with the normal plantlets,hyperhydric plantlets showed abnormal micro structure and ultrastructure.In hyperhydric plantlets,cell wall became thinner,membrane structure was damaged,cytoplasm became rarer,and the number of chloroplasts and starch grains decreased significantly.Conducting tissue was maldeveloped and the cell wall became thin,loose and shrinking,which resulted in collapse and blocking appearing in lumen.The stoma could not close properly when treated with ABA,dark and dehydration.(2)The changes in water and reactive oxygen species(ROS)metabolism in hyperhydric plantlets were investigated.Compared to the normal plantlets,the free water content of hyperhydric plantlets increased by 21%,the bound water content decreased by 18%.the apoplastic water increased by 6.5 times,and the apoplastic air decreased by 89%.The content of hydrogen peroxide(H2O2)and the production rate of oxygen anion radical(O2 -)were increased by 2.2 and 3.1 times,respectively.Moreover,the activities of various antioxidant enzymes,and the contents of ascorbic acid(AsA)and glutathione(GSH)were significantly lower than those in normal plantlets.Furthermore,in comparison with normal plantlets,the content of malondialdehyde(MDA)and relative conductivity increased by 1.5 times and 2.6 times respectively.These results indicated water and ROS metabolism were changed in hyperhydric plantlets,which broken the oxidative balance and caused excessive water accumulation.(3)Whole-genome bisulfate sequencing on normal and hyperhydric seedlings was conducted.We found that the global methylation level(ML)decreased in hyperhydric seedlings compared with normal seedlings,and most of the differentially methylated genes were CHH hypomethylated genes.A total of 4066 significant DMRs,including 1016 hypermethylated and 3050 hypomethylated DMRs,were identified.Hypomethylation was more common in the hyperhydric plants,and the number of DMRs was much greater in the CHH context than in the CHG and CG contexts.The GO enrichment analysis indicated that CHH DMR-related genes and promoters,involved in response to stress,response to stimuli(stress,chemicals and hormones),cellular catabolic processes and the cellular hyperosmotic response pathway,were significantly enriched.These data indicated that DNA demethylation,particularly in the promoter and repeat regions in the CHH context,might be a key regulatory mechanism for HH development.And the genes encoding stress and hormone response-factors may participate in DNA methylation-mediated HH developmental processes.(4)RNA-seq on the normal and hyperhydric seedlings was performed.A total of 2197 transcripts,including 1212 upregulated and 985 downregulated transcripts,were differentially expressed in the hyperhydric seedlings compared with the normal seedlings.The detailed assignments of the DEGs and pathways were determined by MapMan.We noticed that an abundance of DEGs were enriched in the 'ethylene signalling','redox' and 'cell wall'pathways.The correlation between gene transcription level and DNA methylation status in different contexts was further analysed.Gene expression changes were predominantly negatively correlated with CHH MLs in both the body and promoter regions.These results suggest that CHH demethylation may play unique roles in the development of HH morphological characteristics and physiological response processes.(5)ABA-insensitive mutants and wild-type seedlings of Arabidopsis thaliana did exhibit HH symptoms.,while ethylene-insensitive mutants and wild-type seedlings of Arabidopsis thaliana supplemented with AgNO3 cultured on Gelrite media did not develop HH.These results indicated that ethylene plays a vital role in HH development in Arabidopsis.Moreover,the bisulfite sequencing and qPCR results showed that hyperhydric seedlings displayed CHH demethylation patterns in the promoter regions of the ACS1 and ETR1 genes,resulting in upregulated expression of both genes and increased ethylene accumulation.However,the addition of AgNO3 in culture medium could increase the DNA methylation level of ACS1 and ETR1 promoter and decrease the transcription.Excessive ethylene accumulation in hyperhydric seedlings consequently resulted in reducing in stomatal aperture and water loss.Meanwhile,ethylene increased the phosphorylation of aquaporins and water uptake.As a result,excessive amounts of water accumulate in the tissues.However.AgNO3 could decrease the content of endogenous ethylene,increase stomatal aperture,increase water loss rate,decrease the phosphorylation level of aquaporin and water uptake capacity of hyperhydric seedlings.The results showed that the unsuitable culture environment could potentially lead to the demethylation of genes involved in the ethylene pathway.Ethylene synthesis and signal transduction would subsequently increase,which would induce both phosphorylation of aquaporins and stomatal closure,after which water loss would decrease and water uptake would increase.As a result,excessive amounts of water accumulate in the tissues,and HH symptoms occur.On the contrary,AgNO3 prevents HH by blocking the ethylene pathway by maintaining the ML of ethylene signalling pathway genes.(6)About 67%of the hyperhydric Dianthus chinensis L.plantlets were found to revert to normal condition when the plantlets were cultured in medium supplemented with 30 ?mol L-1AgNO3.The hyperhydric rate of the plantlets was reduced from 63.3%to 2.67%by adding 30 ?mol/L AgNO3 to the medium.Physiological and biochemical results showed AgNO3 could reduce the expression of ethylene synthesis and ethylene signal transduction-associated genes,ethylene content,and H2O2 accumulation in guard cells.At the same time,it increased antioxidant enzyme activity,stomatal aperture and water loss,ultimately reducing water content.Taken together,this study demonstrated that DNA demethylation plays a crucial role in hyperhydricity development by activating genes that affect ethylene signalling.In addition,the role of AgNO3 in preventing HH was elucidated in Arabidopsis thaliana,and the effect of AgNO3 in controlling HH was verified in D.chinensis L..These data will be helpful for revealing the epigenetic mechanism that controls HH development and might provide an effective technique for HH control. |
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