Functional Analysis Of PsnNF-YA5 In The Secondary Cell Wall Biosynthesis Of Populus Simonii×populus Nigra

NF-Y5 is a ubiquitous transcription factor of plants in NF-Y family.It plays important roles in the physiological function including embryo,seed development,the drought stressand abscisic acid and so on.Little is known so far about the impact of NF-Y family gene on regulation and biological function of secondary growth.The preliminary studies on biological function and regulatory mechanism of NF-YA5 from Populus simonii×Populus nigra,which participated in the formation of the secondary cell wall.The main contents are as follows.1.NF-YA5 was cloned from Populus simonii×Populus nigra trees in the full length of CDS fragment,it encoded 341 amino acids and had the CBF conservative domain structure in the position of 181-242 and EAR motif-mediated transcriptional repression(Lx Lx Lx)in the position of 187-193,named it Psn NF-YA5.Subcellular localization anal ysis revealed that Psn NF-YA5 was targeted to the cell nucleus.Yeast one hybrid analysis showed that PsnNF-YA5 has no transcription activity.The result has proved that PsnNF-YA5 exerted its biological effects by forming a multi-protein complex with other protein or it might be a transcription repressor.2.Tissue expression profiles indicated PsnNF-YA5 was highest expressioned in secondary tissues.Immunohidtochemical staining indicated that GUS activity existed in root,stem and leaf vein.The result has proved that the Psn NF-YA5 may be involved in the development of plant secondary tissues.3.The results of yeast one hybrid showed that PsnNF-YA5 could recognize the cisacting elements(Box4,GATA,HSE,ACE,G-Box)of the downstream targets(CAD4,CAD9,CCOAMT)of Psn NF-YA5,so as to the cist-acting elements,which were associated with the formation of xylem(ACI,ACII,ACIII).The result indicated that Psn NF-YA5 might participate in the formation of the secondary cell wall through regulating the above-mentioned genes.4.The growth and secondary growth characteristics of transgenic tobacco plants showed that,The height was decreased 28.51%,stem strength was decreased 28.60%,fresh weight and dry weight were decreased 32.24% and 33.89%,respectively;cellulose,hemicelluloses contents was increased 7.7% and 3.3%,respectively,while the lignin content was decreased 37.2%;the cell wall thickness was decreased 22.86%;fiber length was decreased 6.6%,fiber width was increased 13.05%;the genes encoding key enzymes involved in cellulose biosynthetic pathway(Ces A4,Ces A7,Ces A8)and hemicelluloses biosynthetic functional pathways(IRX8,IRX9)were up-regulated,FRA8 was down-regulated,the genes encoding key enzymes involved in lignin biosynthetic pathway(PAL,C4 H,HCT,C3H1,CCOAMT,CCR1,COMT,CAD6,CAD4)were down-regulated obviously,the genes encoding key enzymes involved in cell expansion(SCPL45,SCPL49,XTH5,EXPASIO NA15)were up-regulated,the genes encoding key enzymes involved in programmed cell deathfiber length(XCP1)was down-regulated,while,LBD30 and XND1 were up-regulated.These results showed that Psn NF-YA5 involved in the formation of plant secondary cell wall.5.The analysis of Arabidopsis mutants nf-ya5 indicated that the lignin content was increased,while the cellulose and hemicelluloses contents were decreased in mutants;the cell wall thickness was increased 76.4%;the genes encoding key enzymes involved in cellulose biosynthetic pathway(Ces A4,Ces A7,Ces A8)and hemicelluloses biosynthetic functional pathways(IRX8,IRX9)were down-regulated,FRA8 was up-regulated,the genes encoding key enzymes involved in lignin biosynthetic pathway(PAL,C4 H,HCT,C3H1,CCOAMTCCR1,COMT,CAD6,CAD4)were up-regulated,the genes encoding key enzy mes involved in cell expansion(SCPL45,SCPL49,XTH5,EXPASIONA15)were down-reg ulated,the genes encoding key enzymes involved in programmed cell deathfiber length(XCP1)was up-regulated,while,XND1 and LBD30 were down-regulated.These results preliminarily showed that At NF-YA5 might involved in the formation of plant secondary cell wall.