Functional Characterization Of Histone H3K4 Methyltransferase ATX1 In Secondary Wall Biosynthesis Of Arabidopsis Thaliana

To date,250,000 to 300,000 species of vascular plants has been found widely distributed all over the world.The emergence of vascular tissue in plants improves the efficiency of water transport,which is one of the important reasons why plants can adapt to terrestrial environments.The thickening of the secondary wall of the sclerenchyma cells in vascular plants provides mechanical support and water transport capacity for them,which helps plants to grow upright and adapt to arid environment.Thus,the understanding of secondary wall biosynthesis and regulation mechanism of vascular plants is of great significance for elucidation of the phylogenetic evolution of plantsPrevious studies have shown that in vascular plants,the thickening of secondary wall is finely regulated by a transcriptional regulatory network composed of transcription factors such as NAC(NAM,ATAF,and CUC)and MYB.Although the transcriptional regulation mechanism of secondary wall synthesis in vascular plants has been widely reported,the detailed regulation mechanism of NAC master switch genes during stem development is still unclear.Studies in eucalyptus showed that SND1,a key transcriptional regulator of secondary wall biosynthesis,was enriched in H3K4me3(Histone H3 lysine 4 trimethylation)in the developing xylem^[1],suggesting that H3K4me3 may be involved in the regulation of secondary wall biosynthesis.However,how the H3K4me3 modification at the NAC master gene is established and regulated during the stem development remains unclear.In this study,transcriptome sequencing and Chip-Seq were used to analyze the relationship between H3K4me3 modification and gene expression in Arabidopsis thaliana during the stem development.The molecular biology and genetic methods were used to illustrate the mechanism of H3K4me3 modification in regulation of secondary wall synthesis during the stem development in Arabidopsis.The main results are as follows:1)Representative stages identification of vascular tissue development in Arabidopsis inflorescence stemDuring the stem development of Arabidopsis,the most obvious changes were seen in fiber cells.According to the differentiation of fiber cells and the synthesis of secondary wall,the development of vascular tissue of the stem growing to 20 cm is divided into four stages from the top to the base.At stage I(0-3 cm),only discrete vascular bundles were observed and the fibers have not differentiated yet.At stage II(5-7 cm),fibers begin to differentiate couple with the synthesis of secondary wall.At stage III(10-12 cm),the differentiation of fiber has been almost completed and formed a complete ring,and the secondary wall thickening is vigorous.At stage IV(18-20 cm),the thickening of the secondary wall of fibers has been almost completed.2)Genome-wide expression analysis of Arabidopsis during the stem developmentTo analyze the genome-wide expression patterns during Arabidopsis stem development,transcriptome sequencing was performed with the stem from stage I to III.The Expression patterns of genes were divided into 8 categories through the Short Time-Series Expression Miner Software(STEM).3438 genes were up-regulated and 4238 genes were down-regulated in the early development stage(stage II)compared with stage I.however,only 754 and 727 genes were up-regulated and down-regulated from stage III,respectively.The results of transcriptional sequencing and anatomical analysis showed that the transition from stage I to II is important for the initiation of fiber differentiation and secondary wall synthesis.3)Correlation analysis of H3K4me3 modification level and gene expression level during stem development in ArabidopsisCh IP-seq were used to analyze the genome-wide level of H3K4me3 modification during the stem development(from stage I to II).From stage I to II,a large number of genes were hypomethylated,while 1042 genes were hypermethylated.Among the hypermethylated genes,55%of them are also up-regulated in the expression level,but only0.04%of them were down-regulated.Further analysis of the up-regulated and hypermethylated genes during the stem development through GO(Gene Onotology)revealed significant enrichment in secondary wall synthesis related pathyway,including key regulators of secondary wall synthesis,such as NST1,SND1,SND2,and MYB20,etc.This suggests that H3K4me3 modification may promote the expression of genes related to secondary wall biosynthesis during the stem development of Arabidopsis.4)ATX1 is mainly expressed in interfascicular fiberTo investigate how H3K4me3 modification is regulated during stem development,the expression patterns of the Trx group were detected through q RT-PCR and GUS staining.ATX1 was found up-regulated early during the stem development and mainly expressed in interfascicular fiber from stage II to IV.While other members of Trx group were not specifically expressed in xylem cells.5)ATX1 positively regulates the secondary wall synthesis of interfascicular fiberAnalysis of mutants of Trx group showed that atx1 mutants were more prone to lodging than the wild type.Anatomical analysis showed that the mutation of ATX1 inhibited the secondary wall thickening of interfascicular fiber,and the recovery lines of ATX1restored the secondary wall thickness of interfascicular fiber to the level of WT,while overexpression of ATX1 increased the secondary wall thickness of fiber.The contents of lignin,cellulose and hemicellulose of the secondary wall were significantly decreased in atx1-2,while the overexpression of ATX1 promoted the accumulation of lignin,cellulose and hemicellulose.The results of q RT-PCR also showed that the mutation of ATX1 inhibited the expression of lignin,celllulose and xylan biosynthesis genes.6)ATX1 binds and regulates H3K4me3 modification and expression of NST1 and SND1RNA-seq and H3K4me3 Ch IP-seq revealed that 107 genes were significantly hypomethylated and down-regulated in atx1 mutant.Further analysis found that 26 of the107 genes were hypermethylated and up-regulated during the stem development,including the NAC master switches NST1 and SND1 for secondary wall biosynthesis of fiber.Ch IP-q PCR showed that ATX1 could bind to the loci of NST1 and SND1.7)Regulation of NST1 and SND1 by ATX1 during the stem developmentq RT-PCR and Ch IP-q PCR were used to analyze the changes of H3K4me3 modification and expression level of NST1 and SND1 during stem development.The loci of NST1 and SND1 were hypermethylated with H3K4me3 and up-regulated significantly at stage II and stage III compared with stage I in WT,while the up-regulation of H3K4me3 and expression level of NST1 and SND1 were significantly inhibited in atx1 during the stem development.8)ATX1 regulates the synthesis of secondary wall of fiber mainly through NST1and SND1Previous studies have shown that snd1 nst1 doble mutant resuted in defect of secondary wall of fiber.To verify whether ATX1 regulates the synthesis of secondary wall through NST1 and SND1,we overexpressed ATX1 in snd1 nst1 double mutants.Anatomical observation found that overexpression of ATX1 in WT could promote the synthesis of secondary wall.However,overexpression of ATX1 in snd1 nst1 could not restore the biosynthesis of secondary wall in fiber.The results suggest that ATX1 promotes secondary wall biosynthesis depends on the functional integrity of NST1 and SND1.Genetic experiments showed that the complementation of NST1 and SND1 in atx1-2 could restore the thickness of the secondary wall of interfascular fiber in atx1-2 to the level of WT.In conclusion,Our results suggest that H3K4me3 modification is involved in the regulation of secondary wall synthesis during Arabidopsis stem development.And,ATX1,an H34Kme3 methyltransferase,regulates the synthesis of interfascicular fiber secondary wall by changing the H3K4me3 modification of NST1 and SND1 and promoting their expression during the stem development of Arabidopsis.