A Preliminary Study On Histone Methyltransferase Gene PtrATX1 In Response To Drought Stress In Populus Trichocarpa

Drought is an important environmental limiting factor affecting tree growth.Under drought stress,trees respond to water loss through a series of precise and complex regulatory processes.Epigenetic regulation plays an important role in this process.Trimethylation of lysine 4 on histone H3 protein subunit(H3K4me3)is a typical epigenetic modification marker catalyzed by the histone methyltransferase ATX,which is involved in the regulation of plant responses to stress.In this study,the function of histone methyltransferase PtrATX1 in drought stress response of Populus trichocarpa was studied.According to the genomic information of Arabidopsis thaliana,eight ATX genes were identified in this study,including one PtrATX1,two Ptr ATX2,two Ptr ATX3,one Ptr ATX4 and two Ptr ATX5.Evolutionary analysis showed that PtrATX1 belongs to a separate branch,and the gene may have a unique biological function.RNA-seq and RT-q PCR results showed that PtrATX1 gene was up-regulated in response to drought stress.In order to analyze the biological function of this gene,atx1 mutant was created by CRISPR/Cas9 system.Western blot analysis indicated that the function of PtrATX1 gene was lost and modification level of H3K4me3 was decreased significantly in the atx1 mutant.Phenotypic analysis of atx1 mutant and wild-type P.trichocarpa showed that the loss of PtrATX1 function affected the growth of plant,including an increase in plant height and stem basal diameter,and a decrease in number of stem internodes.The morphological observation by paraffin section and scanning electron microscope showed that there were no significant differences in the morphology,size and number of stem cambium cells between atx1 mutant and wild type.Compared with wild-type plants,the stem of atx1 mutant had fewer vessel and fiber cells with thicker cell wall,large and longer vessel cells,and shorter fiber cells.Phloroglucinol staining of xylem sections showed that lignin deposition level was higher in xylem of atx1 mutants.Analysis of wood components showed that the acid insoluble lignin content and total lignin content of mutants were increased significantly,but there were no significant differences in acid soluble lignin content.The contents of two lignin monomers(S,G)and the ratio of S/G,the content of H-type lignin decreased significantly.Glucose,xylose,galactose,arabinose and total carbohydrate contents did not change in the mutant.Vessel cells are important for water transport in plants.Changes in the number,size and length of vessel cells affect water transport efficiency and drought adaptation of plants.In order to analyze the effects of PtrATX1 mutation on tree adaptation to drought stress,the atx1 mutant and wild-type P.trichocarpa were subjected to drought stress,and the results showed that the atx1 mutant plants exhibited drought-sensitive phenotypes.In conclusion,the loss of the function of PtrATX1 changed the morphological structure of vessel and fiber cells by modifying the deposition mode of lignin in xylem,leading to larger and longer vessel cells.Under drought stress,larger lumen area of vessels increases the risk of air cavitation and embolism,while the increased vessel length is easy to cause the diffusion of air cavitation and embolism,reducing drought tolerance of plants.In addition,the more biomass of atx1 mutant increased the demands of plants for water,which was not conducive to the adaptation to drought stress.In conclusion,histone methyltransferase PtrATX1 plays an important role in drought stress responses.