Molecular Mechanism Of Regulation Of Drought Response And Adaptation By Histone Acetylation In Potato

Potato(Solanum tuberosum L.),the most important tuber food crop,is also a drought-sensitive crop,and drought has a huge impact on potato production.Preliminary laboratory studies have shown that potato plants become more droughttolerant after drought-rehydration cycles,a phenomenon that has also been described as drought memory.A number of studies have shown that histone acetylation and deacetylation modifications play an important role in response and adaptation of plants to abiotic stresses,but the molecular mechanisms of involvement of histone acetylation and deacetylation in the formation of drought memory of potato have not been reported yet.Based on the whole-transcriptome sequencing completed in our laboratory,the present study identified the potato histone acetyltransferase and deacetylase gene families and their corresponding miRNAs involved in the regulation of histone acetylation and deacetylation at the genome-wide level,investigated their possible roles in drought adaptation in potato,and performed cloning and preliminary functional validation of the high drought stress-responsive genes in the two families,with the following main results.1.Identification and functional analysis of potato histone deacetylase family members and their corresponding miRNAsA total of 18 members of the StHDAC gene family were identified from potato,which could be divided into three subfamilies and localized on nine chromosomes,where there were three pairs of fragment replication events,encoding 129 aa～655 aa,with molecular weights ranging from 14161.26 Da～73866.89 Da and theoretical isoelectric points ranging from p I 4.63～9.15.The subcellular localization results showed that StSRT1401 and StSRT1402 were localized in chloroplasts,and the rest were all localized in the nucleus.Cis-acting elements were identified in the promoter sequences of StHDACs gene family members containing a large number of stress response elements.18 members of the family differentially expressed significantly in different organs of potato,as well as under drought,salt and high temperature stresses and during drought-rehydration cycle.847 miRNAs were identified to target and regulate 18 members of the StHDAC gene family,and the miRNAs showed positive and then negative regulation of gene expression with their target StHDACs,indicating that miRNAs are involved in the regulation of gene expression of StHDACs during drought-rehydration cycle.The cloning of the screened StHDT1402 gene was carried out and the results from genetic transformation of yeast showed that the transgenic yeast strains grew significantly better than the control under 5%,10% and 15% PEG6000 stress,and also had stronger tolerance under 10% salt stress compared to the control,indicating that the StHDT1402 gene plays some roles in improving osmotic and salt stress tolerance in yeast in vivo.The overexpression experiment of potato StHDT1402 gene has obtained and identified four diploid potato positive lines(StHDT1402-2,StHDT1402-4,StHDT1402-5,StHDT1402-9),which were verified by RT-q PCR,and the results showed that the gene expression of overexpressed plants was much higher than in the control.2.Identification and functional analysis of potato histone acetyltransferase family members and their corresponding miRNAsA total of 20 histone acetyltransferase genes were identified in the potato genome,divided into four subfamilies,localized on 11 chromosomes.,with the largest numbers in GNAT subfamily.The 20 family members encode 148 aa～1855 aa,with molecular weights ranging from 17147.01 Da～210348.51 Da and theoretical isoelectric points ranging from p I 5.31～9.66.The results of conserved structural domain,Motif and protein structural domain analysis indicated that each subfamily member had conserved acetylated functional structural domains.Digital expression profiling showed that potato histone acetyltransferases were expressed in leaves,stems,tubers,flowers and fruits and were also responsive to abiotic stresses such as salt,high temperature and drought as well as different hormone treatments such as ABA,IAA and GA3.Prediction miRNAs targeted to HAT gene family found that 1098 miRNAs were identified targeting to 20 HAT family members.miR5248-x and miR7980-x were targeted StHAF and StHAG1-5 genes,respectively,and co-expression analysis during drought-rehydration cycle possibly showed a positive correlation trend between m RNAs and miRNAs,indicating that coordination of the HAT genes together with their corresponding miRNAs,regulate the response and adaptation of potato to drought stress.In this study,a candidate gene StHAG3 with the highest differential expression under drought stress was screened from this family and subjected to gene cloning,yeast heterologous expression verification and potato genetic transformation analysis.The results showed that overexpression of StHAG3 gene could improve the tolerance of yeast strains to salt and drought stresses.The overexpression experiment of potato StHAG3 gene has obtained and identified seven diploid potato positive lines(StHAG3-12、StHAG3-21、StHAG3-32、StHAG3-54、StHAG3-67、StHAG3-73、StHAG3-78).The results from Western blot of H3 histone protein showed that H3 acetylation level under drought was higher than that after rehydration in potato during each round of drought-rehydration cycle,indicating that the alteration of total H3 histone acetylation level was involved in response of potato plants to drought stress.The above findings are helpful for further elucidating how the StHDAC and StHAT gene families and their corresponding miRNAs were involved in regulation of drought memory and their drought resistance in potato by regulating histone acetylation and deacetylation,which will lay the foundation for a better understanding of the molecular mechanisms of drought resistance and precise molecular breeding for drought resistance in potato.