The Mutation(Lys To Met) Of Histone H3K4 Influences ABA Response And The Development Of Seedling In Arabidopsis Thaliana

The modification of the histone H3 in the lysine(Lys,K)residue mainly occured on the fourth(K4),ninth(K9),twenty-seventh(K27)and thirty-sixth(K36),which generating three forms methylation: monomethylation,demethylation and trimethylation.In higher plants,histone H3 methylation affects growth and development through regulating gene expression.In general,the growth of higher plants is strongly influenced by a series of abiotic and biotic stresses,such as drought,cold,salt,etc.The modifications of histone H3 play important roles in regulating of plants to adverse environmental cues.Therefore,it is very important to elusive the critical roles of histone H3 modification in the regulation of plant's growth and development,as well as the adaption to the fluctuation of environmental conditions.It has been reported that the mutation of lysine to methionine(Methionine,Met,M)(K-to-M)in histone H3 leads to the decrease of H3 methylation at the whole genome level and the changes of genes expression,which finally influencing the growth and development of plants.To investigate the roles of histone H3 K-M in Arabidopsis thaliana,we generated a point mutation through replacing the fourth Lys(K4)with Met on HTR5(At4g40040),which encodes histone H3.After recombinating the sequence to an overexpression construct,we transformed it into wild type Arabidopsis.The positive lines of H3K4 M overexpression(K4MOEs)were identified by q RT-PCR.The results demonstrated that the expression of H3K4 M is upregulateded in K4MOE-1 and K4MOE-20 compared with wild type.The T3 generation homozygous plants showed strong developmental defects,such as stunting,dwarf with miniature leaf,short silique and partial infertility pollen.In addition,we treated the seedlings of K4 MOE with Na Cl,Mannitol and ABA,respectively,and found that K4 MOE is sensitive to ABA,but not to Na Cl or Mannitol.To further verify the ABA sensitive phenotype of K4 MOE,ABA was used to treat the leaves of WT and K4 MOE,we found that the stomatal closure of K4 MOE were faster.The spatial expression profile of HTR5 was analyzed by GUS staining.The expression of HTR5 was mainly observed in the roots,hypocotyls,veins and shoot meristem,and can be induced by ABA.In addition,we also examined the accumulation of H3K4me1,H3K4me2 and H3K4me3 in K4 MOE by western blot analysis.Compared with wild type,the results showed that the levels of H3K4me2 and H3K4me3 in K4 MOE were significantly decreased,while H3K4me1 was not significantly changed.In parallel,we analyzed the expression of H3K4 methylation targeted gene in ABA synthesis and signal pathway,and found that that the expression of NCED3,ABA3,HB7 were significantly down-regulated.These results suggested that the changes of histone H3K4 methylation could affect the expression of critical genes in ABA synthesis and signalling.In summary,our results provide convincing evidences that the mutation of Lys to Met in histone H3 affects the growth and development of Arabidopsis,and changes the response to ABA by affecting the expression of critical genes that involved in ABA synthesis and signallling.