HDT1 Histone Deacetylase And MYB221 Transcription Factors Regulate The Cambium And Xylem Development

The vascular cambium in plants is characterized by multipotent stem cell identity,which can proliferate and differentiate continuously and is the precondition for root and stem elongation and thickening.The wood is formed by xylem secondary growth,lignin accumulation,cell wall deposition.The HD2 family is identified as a plant specific histone deacetylase,which plays an important role in regulating plant totipotency and is likely to participate in cell proliferation and differentiation.As a class of transcription factors,MYB is widely involved in lignin and cell wall synthesis in xylem.To understand the mechanism of histone deacetylase and transcription factors in regulating xylem development,Arabidopsis thaliana and Populus tomentosa were used as materials for study in this paper.First,transcriptome analysis was performed to detect the genes expression difference in poplar suspension cells which were treated by histone deacetylase inhibitor TSA.Second,the function of HDT1 involved in cambium proliferation,differentiation and xylem development was investigated using hdtl mutants in Arabidopsis.Third,the molecular mechanism of MYB221 involved in xylem development and lignin biosynthesis was analyzed in Populus tomentosa.The results are as follows:Compared with the wild type,cells shape was altered and the acetylation level was increased at H3K9 and H3K27 sites in Populus tomentosa suspension cells treated by 0.5 ?mol/L TSA for 10 days.Transcriptome analysis revealed that 4 465 genes were significantly changed in TSA-treated suspension cells compare with control cells,including 2 363 up-regulated genes and 2 102 down-regulated genes,which related with cell cycle,phenylpropanoid biosynthesis,cell wall components and acetylation related pathways.Eleven WRKY transcription factors and eleven MYB transcription factors were up-regulated.Among them,MYB87 regulated cell wall composition,MYB59 was related to cell division and MYB29 regulated flavonoid biosynthesis.Five IAA transcription factors and three MYB transcription factors were down-regulated,they included MYB85,MYB101,MYB221,which were related to lignin biosynthesis,and IAA8.These results suggested that TSA inhibits the activity of histone deacetylase,which directly or indirectly affects genes related to cell proliferation,cell wall components and lignin synthesis pathway,thus influence cell growth.Semi quantitative PCR was performed to detect HDAC expression in different tissues of Arabidopsis and HDT1 was found highly expressed in stems.Using Arabidopsis hdtl mutants as experimental materials,hdtl mutant plants were significantly weaker than wild type Arabidopsis thaliana during the whole growth process.Meanwhile,paraffin sections showed that the cross sectional area of hdtl mutants was reduced by 34.92-38.10%than that of wild type Arabidopsis.Transmission electron microscopy showed that the xylem number of hdtl mutants was 21.25-22.50%more than that of wild type and the thickness of fiber cells wall in hdtl mutants increased by 22.48-29.36%(P<0.05);the lignin content of hdtl mutant increased by 22.32-27.09%than that of wild type.Transcriptome analysis showed that 127 genes were up-regulated while 45 genes were down-regulated.These include genes involved in auxin signaling(IAA2 and IAA19),lignin biosynthesis(PALI,LAC8 and LAC17)and cellulose biosynthesis(CESA gene family).These ressults indicated that the deletion of HDT1 gene resulted in the thinning of stems,increased number of wood fiber cells,and increased cell wall thickness and lignin content,which suggested that HDT1 directly or indirectly affects the cambium and xylem development.MYB family was found in previous transcriptome analysis.MYB221 was closed to AtMYB4 in evolutional relationship that involved in lignin synthesis.MYB221 was a R2R3-MYB protein and the genomic sequence of MYB221 was 1 638 bp,encoded 269 amino acids,molecular weight was 29 854 Da and pI was 8.67.MYB221 was highly expressed in stems by qPCR detected.The sense and antisense MYB221 was cloned and connected with the pBI121 expression vectors and transformated into Populus tomentosa mediated by Agrobacterium.Two sense transgenic lines(MYB221Z1,MYB221Z12)and one antisense transgenic line(MYB221F1)were identified.The growth of sense MYB221 transgenic plants was not altered while the growth of antisense MYB221 transgenic plants was severely arrested.The lignin content of sense MYB221 transgenic lines in the roots,stems and leaves was higher than that of the wild type,and the difference of lignin content in the stems was more obvious.Comparing with wild type,the lignin content in MYB221Z1 stems was increased by 23.55%,and the lignin content in MYB221Z12 stems increased by 16.73%.The paraffin section showed that the xylem area of the sense MYB221 plant increased by 101.82-133.66%,and the number of tracheary elements increased by 36.54-48.08%.These results implied that MYB221 transcription factors could participate in xylem formation.The above results indicated that both HDT1 and MYB221 transcription factor are involved in xylem development.HDT1 may affect xylem development by directly or indirectly regulating the expression of MYB and xylem related genes.