Isolation And Characterization Of The Poplar PtoMYB175 Transcription Factor Involved In Secondary Cell Wall Formation

As an important wood resource, constructional material, as well as raw material for paper and forage production, Populus has drawn great attention of plant research in the past several decades. And so far, new technologies such as genetical modification have emerged in addition to conventional breeding methods.But in the paper production industry, removal of lignin remains a critical issue and great challenge, it costs so much. Lower lignin content in the raw material and easier means of lignin removal could both bring about significant economical effect, making plant secondary wall biosynthesis pathway a research focus.The MYB family transcription factors, in particular R2R3 MYB transcription factors have been shown to play crucial roles in the regulation of plant secondary cell wall biosynthesis. Our study has shown that PtoMYB175, a MYB transcription factor in Populus tomentosa belongs to the R2R3 subfamily as indicated by sequence alignment, and its homologue in Arabidopsis has been shown as involved in secondary cell wall biosynthesis. However, the specific mechanism remains mysterious on how PtoMYB175 is involved in secondary cell wall biosynthesis. Therefore, we carried out a series of experiments to elucidate the role of PtoMYB175 in secondary cell wall biosynthesis.Expression profiling showed that PtoMYB175 is highly expressed in root and stem, and expressed at a lower level in young leaves and xylem. Subcellular localization analysis revealed that PtoMYB175 is a transcription factor localized in the nucleus. And Yeast one hybrid analysis showed that PtoMYB175 is a transcription repressor. Transgenic poplar strains overexpressing PtoMYB175 showed plural phenotypical abnormality compared to wild type plants, including decreased whole plant height, shorter internode length, decreased stem diameter, and inwardly curled leaves with increased trichomes, which resembled the phenotypes of plants with altered secondary cell wall development.Phloroglucinol staining of plants overexpressing PtoMYB175 showed decreased lignin deposition, as well as lignin autofluorescence detection. Further, toluidine blue staining indicated decreased secondary wall extent and secondary wall cell layers in the overexpression strains, and scanning EM observation revealed fragmentation in the phloem. Moreover, cell wall component determination also revealed decreased lignin, cellulose and xylan contents in the overexpression plants.In addition, q RT-PCR analysis showed that most of the genes encoding key enzymes involved in the lignin, cellulose and xylan biosynthesis pathways, i.e. PAL, C3H3, 4CL5, C4H2, CAD1, CCo Ao MT1, COMT2, Ces A8 and GT43 B were down-regulated in the plants overexpressing PtoMYB175. Such down regulation was further confirmed by transient co-expression of PtoMYB175 and GUS reporter construct containing the promoters of Ptr CCo Ao MT1, Ptr Ces A3 A, Ptr C3 H and Ptr GT43 B.To sum up, our study has shown that PtoMYB175 is a key transcription regulator in the secondary wall biosynthesis pathway of P. tomentosa, and it could repress the expression of several genes encoding key enzymes involved in the pathway, thereby repressing secondary wall biosynthesis in P. tomentosa.